Novel polysaccharide and uses thereof

ABSTRACT

Provided herein is an  E. coli  O polysaccharide, O25B. Also provided herein are prokaryotic host cells containing enzymes (e.g., glycosyltransferases) used in O25B production. The host cells provided herein produce O25B bioconjugates, wherein said bioconjugates contain O25B linked to a carrier protein. Further provided herein are compositions, e.g., pharmaceutical compositions, including O25B and/or bioconjugates containing O25B. Such compositions can be used as vaccines against infection with ExPEC, and may further include one or more additional bioconjugates.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/643,788, filed Jul. 7, 2017, which is a continuation of U.S.application Ser. No. 14/628,844, filed on Feb. 23, 2015, now U.S. Pat.No. 9,700,612, issued Jul. 11, 2017, which claims priority pursuant to35 U.S.C. § 119(e) to U.S. Provisional Application No. 61/943,710, filedon Feb. 24, 2014, the disclosures of which are herein incorporated byreference in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application contains a sequence listing, which is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “SequenceListing_689114-1,” creation date of Oct. 11, 2019,and having a size of 34.9 kb. The sequence listing submitted via EFS-Webis part of the specification and is herein incorporated by reference inits entirety.

1. INTRODUCTION

Disclosed herein are the structure of the E. coli antigen O25B, as wellas uses of O25B, methods of making of O25B, and bioconjugates comprisingO25B. Applicants have identified the E. coli gene cluster responsiblefor production of O25B and have fully characterized the structure of theO25B antigen. Accordingly, provided herein are nucleic acids capable ofproducing O25B in host cells. Also provided herein are host cells, e.g.,recombinantly engineered host cells, comprising nucleic acids capable ofO25B production. Such host cells can be used to generate bioconjugatescomprising O25B linked to a carrier protein, which can be used in, e.g.,the formulation of therapeutics (e.g., vaccines). The O25B antigendescribed herein also is useful in the generation of antibodies, whichcan be used, e.g., in therapeutic methods such as passive immunizationof subjects. Further provided herein are compositions comprising O25B,alone or in combination with other E. coli antigens (e.g., O1, O2, andO6 and subserotypes thereof), for use in therapeutic methods, e.g.,vaccination of hosts against infection with E. coli (e.g.,extra-intestinal pathogenic, such as uropathogenic, E. coli).

2. BACKGROUND

Extra-intestinal pathogenic E. coli (ExPEC) causes a wide variety ofinfections that are responsible for significant morbidity, mortality,and costs annually. Urinary tract infections are among the most frequentconditions caused by ExPEC in human beings. However, life-threateningconditions, such as meningitis and sepsis, also are caused by ExPEC.

Bacterial resistance to antibiotics is a major concern in the fightagainst bacterial infection, and multi-drug resistant (MDR) E. colistrains are becoming more and more prevalent. Schito et al., 2009, Int.J. Antimicrob. Agents 34(5):407-413; and Pitout et al., 2012, ExpertRev. Anti. Infect. Ther. 10(10): 1165-1176. Thus, the development ofefficient vaccines against ExPEC is needed.

3. SUMMARY

In one aspect, provided herein is a prokaryotic host cell comprisingnucleic acids encoding enzymes (e.g., glycosyltransferases) capable ofproducing the novel polysaccharide disclosed herein, E. coli O25B. Alsoprovided herein are host cells comprising nucleic acids encoding enzymes(e.g., glycosyltransferases) capable of producing other E. coliantigens, e.g., O25A, O1, O2, and O6, and subserotypes thereof. The hostcells provided herein may naturally express nucleic acids specific forproduction of an O antigen of interest, or the host cells may be made toexpress such nucleic acids, i.e., in certain embodiments said nucleicacids are heterologous to the host cells. In certain embodiments, thehost cells provided herein comprise nucleic acids encoding additionalenzymes active in the N-glycosylation of proteins, e.g., the host cellprovided herein can further comprise a nucleic acid encoding anoligosaccharyl transferase or nucleic acids encoding otherglycosyltransferases. In certain embodiments, the host cells providedherein comprise a nucleic acid encoding a carrier protein, e.g., aprotein to which oligosaccharides and/or polysaccharides can be attachedto form a bioconjugate. In a specific embodiment, the host cell is E.coli. See Section 5.3.

In a specific embodiment, provided herein is a prokaryotic host cellcomprising an E. coli rfb(upec138) gene cluster (SEQ ID NO:12), or agene cluster that is about or at least 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identical to or homologous to an E. coli rfb(upec138)gene cluster (SEQ ID NO:12). In a specific embodiment, the prokaryotichost cell comprises a nucleic acid sequence encoding an oligosaccharyltransferase. In another specific embodiment, the prokaryotic host cellfurther comprises a nucleic acid sequence encoding a carrier proteincomprising a consensus sequence Asn-X-Ser(Thr), wherein X can be anyamino acid except Pro (SEQ ID NO:14); or a carrier protein comprising aconsensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z areindependently selected from any natural amino acid except Pro (SEQ IDNO:15) (see WO 2006/119987). In a specific embodiment, the host cell isE. coli.

In another specific embodiment, provided herein is a prokaryotic hostcell comprising an E. coli rjb(upec163) gene cluster, or a gene clusterthat is about or at least 75%, 80/%, 85%, 90%, 95%, 96%, 97%, 98%, or99% identical to or homologous to an E. coli rfb(upec163) gene cluster.In a specific embodiment, the prokaryotic host cell comprises a nucleicacid sequence encoding an oligosaccharyl transferase. In anotherspecific embodiment, the prokaryotic host cell further comprises anucleic acid sequence encoding a carrier protein comprising a consensussequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQID NO: 14); or a carrier protein comprising a consensus sequenceAsp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selectedfrom any natural amino acid except Pro (SEQ ID NO: 15) (see WO2006/119987). In a specific embodiment, the host cell is E. coli.

In another specific embodiment, provided herein is a prokaryotic hostcell comprising an E. coli rfb(upec177) gene cluster, or a gene clusterthat is about or at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to or homologous to an E. coli rb(upec177) gene cluster. In aspecific embodiment, the prokaryotic host cell comprises a nucleic acidsequence encoding an oligosaccharyl transferase. In another specificembodiment, the prokaryotic host cell further comprises a nucleic acidsequence encoding a carrier protein comprising a consensus sequenceAsn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ IDNO:14); or a carrier protein comprising a consensus sequenceAsp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selectedfrom any natural amino acid except Pro (SEQ ID NO: 15) (see WO2006/119987). In a specific embodiment, the host cell is E. coli.

In another specific embodiment, provided herein is a prokaryotic hostcell recombinantly engineered to comprise (e.g., by introduction of oneor more vectors/plasmids into the host cell) one, two, three, four, ormore of the following genes (or a nucleic acid that is about or at least75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to orhomologous to one of the following genes): rmlB (SEQ ID NO:1), rmlD (SEQID NO:2), rmlA (SEQ ID NO:3), rmlC (SEQ ID NO:4), wzx (SEQ ID NO:5),wekA (SEQ ID NO:6), wekB (SEQ ID NO:7), wzy (SEQ ID NO:8), wbbJ (SEQ IDNO:9), wbbK (SEQ ID NO:10), and/or wbbL (SEQ ID NO: 1). In anotherspecific embodiment, the prokaryotic host cell comprises a nucleic acidsequence encoding an oligosaccharyl transferase (e.g., a heterologousoligosaccharyltransferase). In another specific embodiment, theprokaryotic host cell further comprises a nucleic acid sequence encodinga carrier protein comprising a consensus sequence Asn-X-Ser(Thr),wherein X can be any amino acid except Pro (SEQ ID NO:14); or a carrierprotein comprising a consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr),wherein X and Z are independently selected from any natural amino acidexcept Pro (SEQ ID NO:15) (see WO 2006/119987). In a specificembodiment, the host cell is E. coli.

In another specific embodiment, provided herein is a prokaryotic hostcell recombinantly engineered to comprise (e.g., by introduction of oneor more vectors/plasmids into the host cell) one, two, three, four, ormore of the following (i) dTDP-Glucose 4,6-dehydratase; (ii)dTDP-6-Deoxy-D-glucose 3,5-epimerase; (iii) Glucose-1-phosphatethymidylyltransferase; (iv) dTDP-4-dehydrorhamnose 3,5-epimerase; (v) Oantigen flippase; (vi) dTDP-Rha:Glc-Rha(Ac)-GlcNAc-UPPα-1,3-rhamnosyltransferase; (vii) UDP-Glc:Rha-GlcNAc-UPPα-1,3-glucosyltransferase; (viii) O antigen polymerase; (ix) O-acetyltransferase; (x) UDP-Glc:Rha-GlcNAc-UPP α-1,3-glucosyltransferase;and/or (xi) dTDP-Rha: GIcNAc-UPP α-1,3-rhamnosyltransferase. In aspecific embodiment, the prokaryotic host cell comprises a nucleic acidsequence encoding an oligosaccharyl transferase (e.g., a heterologousoligosaccharyltransferase). In another specific embodiment, theprokaryotic host cell further comprises a nucleic acid sequence encodinga carrier protein comprising a consensus sequence Asn-X-Ser(Thr),wherein X can be any amino acid except Pro (SEQ ID NO:14); or a carrierprotein comprising a consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr),wherein X and Z are independently selected from any natural amino acidexcept Pro (SEQ ID NO:15) (see WO 2006/119987). In a specificembodiment, the host cell is E. coli.

In certain embodiments, the prokaryotic host cells provided hereincomprise a deletion or functional inactivation of one or more genes. SeeSection 5.3.1. In a specific embodiment, one or more of the waaL gene,gtrA gene, gtrB gene, gtrS gene, or the rjb gene cluster (or a gene orgenes in the rjb cluster) is deleted or functionally inactivated fromthe genome of a prokaryotic host cell provided herein.

The carrier proteins expressed by the prokaryotic host cells providedherein can be selected from any carrier proteins known to those of skillin the art, e.g., detoxified Exotoxin A of P. aeruginosa (EPA; see,e.g., Ihssen, et al., (2010) Microbial cell factories 9, 61), CRM 197,maltose binding protein (MBP), Diphtheria toxoid, Tetanus toxoid,detoxified hemolysin A of S. aureus, clumping factor A, clumping factorB, E. coli FimH, E. coli FimHC, E. coli heat labile enterotoxin,detoxified variants of E. coli heat labile enterotoxin, Cholera toxin Bsubunit (CTB), cholera toxin, detoxified variants of cholera toxin, E.coli Sat protein, the passenger domain of E. coli Sat protein,Streptococcus pneumoniae Pneumolysin and detoxified variants thereof, C.jejuni AcrA, and C. jejuni natural glycoproteins. In a specificembodiment, the carrier protein expressed by a prokaryotic host cellprovided herein is detoxified Pseudomonas exotoxin (EPA). In certainembodiments, the carrier protein of a host cell provided hereincomprises a signal sequence for targeting the carrier protein into theperiplasmic space of the host cell. In a specific embodiment, the signalsequence is from E. coli DsbA, E. coli outer membrane porin A (OmpA), E.coli maltose binding protein (MalE), Erwinia carotovorans pectate lyase(PelB), FlgI, NikA, or Bacillus sp. endoxylanase (XynA), heat labile E.coli enterotoxin LTIIb, Bacillus endoxylanase XynA, or E. coli flagellin(FlgI). In certain embodiments, the nucleic acid sequence encoding thecarrier protein expressed by the host cells provided herein has beenengineered (e.g., via recombinant techniques) to encode one or more ofthe consensus sequence Asn-X-Ser(Thr), wherein X can be any amino acidexcept Pro (SEQ ID NO:14); and/or the consensus sequenceAsp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selectedfrom any natural amino acid except Pro (SEQ ID NO:15) (see WO2006/119987). In certain embodiments, the carrier proteins expressed bythe host cells provided herein comprise two, three, four, five or moreof said consensus sequences. See Section 5.3.2.

In another aspect, provided herein is a method of producing anN-glycosylated carrier protein (also referred to herein as abioconjugate) that comprises a carrier protein (e.g., EPA) N-linked toan E. coli O antigen (e.g., E. coli O25B), said method comprisingculturing a host cell described herein under conditions suitable for theproduction of proteins, and purifying the N-glycosylated carrierprotein. Methods for producing proteins using host cells, e.g., E. coli,and isolating proteins produced by host cells, are well-known in theart. See Section 5.3.

In another aspect, provided herein are bioconjugates produced by thehost cells provided herein. In a specific embodiment, provided herein isa bioconjugate comprising a carrier protein (e.g., EPA) N-linked to E.coli O25B. See Section 5.4.

In a specific embodiment, provided herein is a bioconjugate comprising acarrier protein (e.g., EPA) linked to a compound of Formula O25B,presented below:

wherein n is an integer between 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25,20 to 25, 10 to 20, or 15 to 20. In a specific embodiment, the carrierprotein is N-linked to the O antigen of Formula O25B.

In another specific embodiment, provided herein is a bioconjugatecomprising a carrier protein (e.g., EPA) linked to a compound of FormulaO25B′, presented below:

wherein n is an integer between 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25,20 to 25, 10 to 20, or 15 to 20. In a specific embodiment, the carrierprotein is N-linked to the O antigen of Formula O25B′.

In another aspect, provided herein is an isolated O antigen from anExPEC E. coli strain, wherein said strain produces O25B. In anotherspecific embodiment, provided herein is an isolated O antigen from E.coli strain upec138. In a specific embodiment, provided herein is anisolated O antigen from E. coli strain upec163. In another specificembodiment, provided herein is an isolated O antigen from E. coli strainupec177. See Section 5.2.

In another aspect, provided herein is a population of isolatedmacromolecules of the Formula O25B, presented below:

wherein n is an integer between 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25,20 to 25, 10 to 20, or 15 to 20. In a specific embodiment, n of at least80% of the macromolecules in the population is between 1 to 30, 1 to 20,1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20.

In another aspect, provided herein is a population of isolatedmacromolecules of the Formula O25B′, presented below:

wherein n is an integer between 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25,20 to 25, 10 to 20, or 15 to 20. In a specific embodiment, n of at least80% of the macromolecules in the population is between 1 to 30, 1 to 20,1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20.

In another aspect, provided herein are methods of generating anti-O25Bantibodies using O25B and/or a bioconjugate comprising O25B. Furtherprovided herein are antibodies produced according to such methods. SeeSection 5.5.

In another aspect, provided herein are compositions, e.g.,pharmaceutical compositions, comprising the bioconjugates providedherein and/or the macromolecules (or populations thereof) providedherein. See Section 5.6.

In a specific embodiment, provided herein is a composition, e.g., apharmaceutical composition, comprising a macromolecule comprising astructure of Formula O25B:

wherein n is an integer between 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25,20 to 25, 10 to 20, or 15 to 20.

In another specific embodiment, provided herein is a composition, e.g.,a pharmaceutical composition, comprising a macromolecule comprising astructure of Formula O25B′:

wherein n is an integer between 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25,20 to 25, 10 to 20, or 15 to 20.

In another specific embodiment, provided herein is a composition, e.g.,a pharmaceutical composition, comprising a bioconjugate describedherein, wherein said bioconjugate comprises a carrier protein (e.g.,EPA) linked to a compound of Formula O25B, presented below:

wherein n is an integer between 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25,20 to 25, 10 to 20, or 15 to 20. In a specific embodiment, the carrierprotein is N-linked to the O antigen of Formula O25B′.

In another specific embodiment, provided herein is a composition, e.g.,a pharmaceutical composition, comprising a bioconjugate describedherein, wherein said bioconjugate comprises a carrier protein (e.g.,EPA) linked to a compound of Formula O25B′, presented below:

wherein n is an integer between 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25,20 to 25, 10 to 20, or 15 to 20. In a specific embodiment, the carrierprotein is N-linked to the O antigen of Formula O25B′.

In certain embodiments, the pharmaceutical compositions provided hereincomprise one or more additional E. coli O antigens, wherein saidantigens are not O25B (e.g., the formula O25B or the formula O25B′),e.g., O antigens from E. coli (e.g., ExPEC) other than those from an E.coli O25B serotype, and/or one or more bioconjugates comprising acarrier protein linked to an E. coli O antigen, wherein said antigen isnot O25B (e.g., the formula O25B or the formula O25B′). Suchcompositions may comprise one or more additional macromoleculescomprising an ExPEC O antigen and/or one or more additionalbioconjugates, e.g., an O1A, O2, and/or O6 macromolecule and/or an O1A,O2, and/or O6 bioconjugate.

In a specific embodiment, provided herein is a composition, e.g., apharmaceutical composition, comprising one or more additionalmacromolecules comprising an ExPEC O antigen and/or one or moreadditional bioconjugates, in addition to an O25B macromolecule (e.g., amacromolecule comprising the formula O25B or the formula O25B′) and/oran O25B bioconjugate (e.g., a bioconjugate comprising a carrier proteinlinked to the formula O25B or the formula O25B′), wherein saidadditional macromolecules comprise a structure selected from the groupconsisting of:

In a specific embodiment, provided herein is a composition, e.g., apharmaceutical composition, comprising one, two, three, four, five, six,or seven macromolecules or bioconjugates comprising said macromolecules,in addition to an O25B macromolecule (e.g., a macromolecule comprisingthe formula O25B or the formula O25B′) and/or an O25B bioconjugate(e.g., a bioconjugate comprising a carrier protein linked to the formulaO25B or the formula O25B′), wherein said additional macromoleculescomprise a structure selected from the group consisting of:

In another specific embodiment, provided herein is a composition, e.g.,a pharmaceutical composition, comprising (i) an O25 (e.g., O25A or O25B)macromolecule, or a bioconjugate comprising O25 (e.g., O25A or O25B) and(ii) an O1 macromolecule or a bioconjugate comprising O1. In a specificembodiment, said O25 macromolecule is an O25B macromolecule. In anotherspecific embodiment, said O1 macromolecule is O1A. In another specificembodiment, said O1 macromolecule is O1B. In another specificembodiment, said O1 macromolecule is O1C.

In another specific embodiment, provided herein is a composition, e.g.,a pharmaceutical composition, comprising (i) an O25 (e.g., O25A or O25B)macromolecule, or a bioconjugate comprising O25 (e.g., O25A or O25B) and(ii) an O2 macromolecule or a bioconjugate comprising O2. In a specificembodiment, said O25 macromolecule is an O25B macromolecule.

In another specific embodiment, provided herein is a composition, e.g.,a pharmaceutical composition, comprising (i) an O25 (e.g., O25A or O25B)macromolecule, or a bioconjugate comprising O25 (e.g., O25A or O25B) and(ii) an O6 macromolecule (e.g., an O6 macromolecule comprising abranching Gic monosaccharide (O6Glc) or a branching GlcNAcmonosaccharide (O6GlcNAc)) or a bioconjugate comprising O6. In aspecific embodiment, said 025 macromolecule is an O25B macromolecule. Inanother specific embodiment, said O6 macromolecule is an O6macromolecule comprising a branching Glc monosaccharide (O6Glc).

In another specific embodiment, provided herein is a composition, e.g.,a pharmaceutical composition, comprising at least two of the following:(i) an O25 (e.g., O25A or O25B) macromolecule or a bioconjugatecomprising O25 (e.g., O25A or O25B); (ii) an O1 macromolecule or abioconjugate comprising O1; (iii) an O2 or a bioconjugate comprising O2;and/or (iv) an O6 macromolecule (e.g., a O6 macromolecule comprising abranching Glc monosaccharide or a branching GIcNAc monosaccharide) or abioconjugate comprising O6. In a specific embodiment, said O25macromolecule is an O25B macromolecule. In another specific embodiment,said O1 macromolecule is OIA. In another specific embodiment, said O1macromolecule is OIB. In another specific embodiment, said O1macromolecule is OIC. In another specific embodiment, said O6macromolecule is an O6 macromolecule comprising a branching Glcmonosaccharide (also referred to herein as O6Glc).

In another specific embodiment, provided herein is a composition, e.g.,a pharmaceutical composition, comprising an O25B macromolecule, an OlAmacromolecule, an O2 macromolecule, and an O6 macromolecule comprising abranching Glc monosaccharide. In certain embodiments, saidmacromolecules are conjugated to carrier proteins.

In another aspect, provided herein are methods of preventing infectionof a subject, e.g., a human subject, by ExPEC, comprising administeringto the subject a pharmaceutically effective amount of a composition(e.g., an immunogenic composition) described herein. See Section 5.7.

In another aspect, provided herein are methods of treating infection ofa subject, e.g., a human subject, wherein the subject is infected withExPEC, comprising administering to the subject a pharmaceuticallyeffective amount of a composition (e.g., an immunogenic composition)described herein. See Section 5.7.

In another aspect, provided herein are methods of inducing an immuneresponse against ExPEC in a subject, e.g., a human subject, comprisingadministering to the subject a pharmaceutically effective amount of acomposition (e.g., an immunogenic composition) described herein. SeeSection 5.7.

In another aspect, provided herein are methods of inducing theproduction of opsonophagocytic antibodies against ExPEC in a subject,e.g., a human subject, comprising administering to the subject apharmaceutically effective amount of a composition (e.g., an immunogeniccomposition) described herein. See Section 5.7.

Terms and Abbreviations

OPS: O polysaccharide; the O antigen of Gram-negative bacteria. OPS alsoare referred to herein as O antigen.

rfb cluster: a gene cluster (e.g., an E. coli gene cluster) that encodesenzymatic machinery capable of synthesis of an O antigen backbonestructure. The term rjb cluster may apply to any O antigen biosyntheticcluster, including those from bacteria that do not belong to genusEscherichia.

waaL: the O antigen ligase gene encoding a membrane bound enzyme with anactive site located in the periplasm. The encoded enzyme transfersundecaprenylphosphate (UPP)-bound O antigen to the lipid A core, forminglipopolysaccharide.

wecA: the first gene encoded in the wec cluster. The encoded proteincatalyzes the transfer of a GlcNAc-phosphate from UDP-GlcNAc to UPP toform UPP-bound GIcNAc.

ECA: enterobacterial common antigen.

RU: repeat unit. As used herein, the RU is set equal to the Biologicalrepeat unit, BRU.

The BRU describes the RU of an O antigen as it is synthesized in vivo.

UPP: undecaprenylpyrophosphate.

LLO: lipid linked oligosaccharide.

O2AB: 2 amino benzamide.

MS: mass spectroscopy.

O25B: the term O25B refers to the O25B antigen from E. coli identifiedherein (a subserotype of E. coli serotype O25). Reference to O25B hereinencompasses the formula O25B and the formula O25B′, both identifiedabove.

O25A: the term O25A refers to the O25A antigen of E. coli (a subserotypeof E. coli serotype O25). Reference to O25A herein encompasses theformula O25A and the formula O25A′, both identified above.

O1A: the term O1A refers to the OlA antigen of E. coli (a subserotype ofE. coli serotype O1). Reference to OIA herein encompasses the formulaOlA and the formula O1A′, both identified above.

O1B: the term O1B refers to the O1B antigen of E. coli (a subserotype ofE. coli serotype O1). Reference to O1B herein encompasses the formulaO1B and the formula O1B′, both identified above.

O1C: the term O1C refers to the O1C antigen of E. coli (a subserotype ofE. coli serotype O1). Reference to O1C herein encompasses the formulaO1C and the formula O1C′, both identified above.

O2: the term O2 refers to the O2 antigen of E. coli (E. coli serotypeO2). Reference to O2 herein encompasses the formula O2 and the formulaO2′, both identified above.

O6: the term O6 refers to the O6 antigen of E. coli (E. coli serotypeO6). Reference to O6 herein encompasses the formula O6 and the formulaO6′, both identified above.

Bioconjugate: the term bioconjugate refers to conjugate between aprotein (e.g., a carrier protein) and an antigen, e.g., an O antigen(e.g., O25B) prepared in a host cell background, wherein host cellmachinery links the antigen to the protein (e.g., N-links).Glycoconjugates include bioconjugates, as well as sugar antigen (e.g.,oligo- and polysaccharides)-protein conjugates prepared by other means,e.g., by chemical linkage of the protein and sugar antigen.

The term “about,” when used in conjunction with a number, refers to anynumber within ±1, ±5 or ±10% of the referenced number.

As used herein, the term “effective amount,” in the context ofadministering a therapy (e.g., a composition described herein) to asubject refers to the amount of a therapy which has a prophylacticand/or therapeutic effect(s). In certain embodiments, an “effectiveamount” refers to the amount of a therapy which is sufficient to achieveone, two, three, four, or more of the following effects: (i) reduce orameliorate the severity of an ExPEC infection or symptom associatedtherewith; (ii) reduce the duration of an ExPEC infection or symptomassociated therewith; (iii) prevent the progression of an ExPECinfection or symptom associated therewith; (iv) cause regression of anExPEC infection or symptom associated therewith; (v) prevent thedevelopment or onset of an ExPEC infection, or symptom associatedtherewith; (vi) prevent the recurrence of an ExPEC infection or symptomassociated therewith; (vii) reduce organ failure associated with anExPEC infection; (viii) reduce hospitalization of a subject having anExPEC infection; (ix) reduce hospitalization length of a subject havingan ExPEC infection; (x) increase the survival of a subject with an ExPECinfection; (xi) eliminate an ExPEC infection in a subject; (xii) inhibitor reduce ExPEC replication in a subject; and/or (xiii) enhance orimprove the prophylactic or therapeutic effect(s) of another therapy.

As used herein, the term “in combination,” in the context of theadministration of two or more therapies to a subject, refers to the useof more than one therapy. The use of the term “in combination” does notrestrict the order in which therapies are administered to a subject. Forexample, a first therapy (e.g., a composition described herein) can beadministered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks,5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, orsubsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours,72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks,8 weeks, or 12 weeks after) the administration of a second therapy to asubject.

As used herein, the term “subject” refers to an animal (e.g., birds,reptiles, and mammals). In another embodiment, a subject is a mammalincluding a non-primate (e.g., a camel, donkey, zebra, cow, pig, horse,goat, sheep, cat, dog, rat, and mouse) and a primate (e.g., a monkey,chimpanzee, and a human). In certain embodiments, a subject is anon-human animal. In some embodiments, a subject is a farm animal or pet(e.g., a dog, cat, horse, goat, sheep, pig, donkey, or chicken). Inanother embodiment, a subject is a human. In another embodiment, asubject is a human infant. In another embodiment, a subject is a humanchild. In another embodiment, a subject is a human adult. In anotherembodiment, a subject is an elderly human. In another embodiment, asubject is a premature human infant. The terms “subject” and “patient”may be used herein interchangeably.

As used herein, the term “premature human infant” refers to a humaninfant born at less than 37 weeks of gestational age.

As used herein, the term “human infant” refers to a newborn to 1 yearold human.

As used herein, the term “human toddler” refers to a human that is 1years to 3 years old.

As used herein, the term “human child” refers to a human that is 1 yearto 18 years old.

As used herein, the term “human adult” refers to a human that is 18years or older.

As used herein, the term “elderly human” refers to a human 65 years orolder.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Pathway for O25A biosynthesis. Arrows indicate individualenzymatic conversions, enzyme names are indicated. Nucleotide activatedsugars are prepared in the cytoplasm either by enzymes provided in the Oantigen cluster or by housekeeping enzymes of the Gram-negative hostcell. A glycosylphosphate transferase (WecA) adds D-GlcNAc phosphate toundecaprenyl phosphate (UP), forming GlcNAc-UPP. Specificglycosyltransferases then elongate the UPP-GlcNAc molecule further byadding monosaccharides forming the biological repeat unit (BRU)oligosaccharide (WekABC WbuB). The indicated order of enzymes does notrefer to the sequence of events during BRU synthesis (indicated by < >).The BRU is then flipped into the periplasmic space by Wzx. Wzy linearlypolymerizes periplasmic BRU's to form the O antigen polysaccharide.Polymer length is controlled by Wzz. Many bacterial oligo- andpolysaccharides are assembled on UPP and then transferred to othermolecules, i.e., UPP is a general building platform for oligo- andpolysaccharide in bacteria. In E. coli, and most other gram negativebacteria, the O antigen is transferred from UPP to lipid A core by theE. coli enzyme WaaL to form lipopolysaccharide (LPS).

FIGS. 2A and 2B: rfb cluster, structure, and pathway forwzx/wzy-dependent O-antigen synthesis, exemplified by the E. coli O25Arfb cluster and O antigen. FIG. 2A shows the rfb cluster structure of E.coli strain E47a, located between the galF and gnd genes. Genes areshown as arrows and filling is indicated according to the function ofthe gene products: black are genes for nucleotide-activatedmonosaccharide biosynthesis which are not part of the housekeepingrepertoire of E. coli (those are encoded elsewhere in the genome),black/white diagonal stripes are glycosyltransferases responsible foradding single monosaccharide units to the BRUs, flippase wzx andpolymerase wzy. FIG. 2B shows the chemical structure of the BRU of theO25A O antigen as presented (see Fundin et al., 2003, Magnetic Resonancein Chemistry 41, 4).

FIG. 3A: O25A, O25B, and O16 BRU structures. FIG. 3B: O antigenbiosynthesis cluster (rib cluster) comparison between O25A, O25B, andO16. Black filled genes are genes involved in nucleotide activatedmonosaccharide biosynthesis, diagonal stripes are predictedglycosyltransferase genes, grey filling indicates BRU processing ortransportation genes, and vertical stripes show O-acetyltransferasehomologies. Grey boxes indicate homology scores above 25% between thegenes; detailed values are indicated. Thin black and grey arrows showannealing locations of typing PCR oligonucleotides for wzy (O25A andO25B specific) and the O25B 3′ region (O25B specific).

FIG. 4: Serotype distribution from the epidemiology study. E. coli Oantigen serotypes identified in samples of community acquired UTIspecimens were grouped according to occurrence

FIGS. 5A and 5B: Silver and Western stain analysis of LPS from clinicalisolates with an O25 positive agglutination phenotype. Strain numbersare indicated above the gel lanes. Individual clones were grown and ODnormalized biomass was harvested by centrifugation. Pellets weredissolved in SDS PAGE Lämmli buffer and treated with proteinase K tohydrolyze all proteins in the sample. Standard silver staining wasapplied to the PAGE gel shown in FIG. 5A, and probing of nitrocellulosemembranes containing electrotransferred material from identically rungels with commercial O25 agglutination antiserum is shown in FIG. 5B.

FIG. 6: 2AB HPLC traces of O25A and O25B samples. 2AB labeled LLOsamples from strains upec138 (dotted line) and upec436 (solid line) wereprepared. Peaks were collected and corresponding BRU structures deducedfrom the MS/MS fragmentation pattern detailed in FIG. 7 are indicated byarrows.

FIGS. 7A and 7B: MS/MS fragmentation ion series obtained from peaksindicated in FIG. 6 at 50′ and 62′ elution times of mother ions m/z=1022(FIG. 7A; from strain upec138) or m/z=1021 (FIG. 7B; from upec_436). Ionseries are shown in relation to the cartoon of the putative BRU.

FIG. 8: Deacetylation of 2AB labeled LLO sample derived from an O25Bpositive clinical isolate. The O25B specific peak at 50′ elution timeobtained from 2AB labeled LLOs of a clinical isolate with the O25Bgenotype was collected, and analyzed by normal phase HPLC aftertreatment with (solid line) or without (dotted line) NaOH for hydrolysisof ND Cal's Special Patent Program O-acetyl groups.

FIG. 9: Monosaccharide composition analysis of O25A and O25Bbioconjugates. O25 bioconjugates were produced, purified, and processedfor monosaccharide composition analysis. C18 HPLC traces of samples areshown. O25A (solid) and O25B (dotted) derived samples are compared to amix of monosaccharides from commercial sources (Glc, GlcNAc, Rha,FucNAc). The elution times of the monosaccharides are indicated byarrows.

FIG. 10: Characterization of O25A bioconjugates. Purified final bulk of4S-EPA-O25A bioconjugates was analyzed by SDS PAGE and visualized bydirect Coomassie staining (C) and Western blotting using either anti-EPAantiserum or anti-O25 antiserum.

FIG. 11: Characterization of O25B bioconjugates. Purified final bulk of4S-EPA-O25B bioconjugates was analyzed by SDS PAGE and visualized bydirect Coomassie staining (C) and Western blotting using either anti-EPAantiserum or anti-025 antiserum.

FIGS. 12A and 12B: O1 O antigen genetic biosynthesis and chemicalstructure. FIG. 12A. The rfb cluster and flanking genes of the O1Astrain E. coli G1632 (ACCESSION NO. GU299791) is shown. Black, grey andstriped color codes are the same as those for FIG. 2, described above.FIG. 12B. Chemical BRU structures of O1 subserotypes are shown.

FIGS. 13A and 13B: Analysis of LPS from clinical isolates with an O1positive agglutination phenotype. FIG. 13A. Silver staining and FIG.13B. Western blotting using anti-O1 antiserum.

FIGS. 14A and 14B: Identification of O1A in O1 clinical isolates. 2ABlabeled LLO samples from O1 clinical isolates were analyzed by LLOfingerprinting. FIG. 14A. Normal phase HPLC traces from 60′ onwards areshown. The baseline for every sample was shifted to visualizeco-migrating peaks. The upec number indicates the clinical strain. FIG.14B. MS/MS fragmentation ion series of m/z=1849.6 (Na+ adduct). Thecartoon assigns the fragmentation ion pattern and probable glycosidicbond breakages in an oligosaccharide of 2 BRU of O1A.

FIG. 15: O1 bioconjugates. Small scale expression test of EPA-O1glycoprotein by E. coli cells (W3110 ΔrfbO16::rf1O1 ΔwaaL) transformedwith an EPA expression plasmid (pGVXN659) and five different pglBexpression plasmids: A, p114: expression of non-codon optimized, HA tagcontaining pglB; B, p939: codon optimized, HA tag containing pglB; C,p970: codon optimized, HA tag removed pglB; D, codon optimized, HA tagcontaining, natural glycosylation site N534Q removed pglB; and E, codonoptimized, HA tag removed, natural glycosylation site N534Q removedpglB. Cells were grown and induced with arabinose and IPTG, afterovernight incubation at 37° C., cells were harvested and periplasmicprotein extracts were prepared. Extracts were then separated by SDSPAGE, transferred to nitrocellulose membranes by electroblotting, andimmunodetected using an anti-EPA serum.

FIG. 16: The bioconjugates described in FIG. 15, detected with anti-O1serum.

FIGS. 17A and 17B: O6 genetic and chemical structures. FIG. 17A. Oantigen biosynthesis cluster (rfb cluster) and flanking genes of E. coliCFT073 (Genbank AE014075.1). Putative gene functions according to BLASTare indicated and genes specific for O6 O antigen biosynthesis areindicated. FIG. 17B. Chemical structures of reported O6 BRU structures(Jann et al., Carbohydr. Res. 263 (1994) 217-225).

FIGS. 18A and 18B: Identification of O6 with branching Glc. 2AB labeledLLO samples from O6 clinical isolates were analyzed by LLOfingerprinting. FIG. 18A. Normal phase HPLC traces from 60′ onwards areshown. Extracts were prepared from reference strains CCUG11309 (thinsolid line) and 11311 (dashed) containing Glc and GlcNAC branches. Theoverlay shows clear differences in elution times of the indicated BRUs.FIG. 18B. Extracts from clinical isolates as indicated by upec numberare compared to the reference strains from A.

FIGS. 19A and 19B: O2 O antigen genetic biosynthesis and chemicalstructures. FIG. 19A. O antigen biosynthesis cluster (rib cluster) andflanking genes of strain E. coli G1674 (accession No. GU299792). Black,grey, and striped color codes are as described in previous figures(e.g., FIG. 2). FIG. 19B. Chemical BRU structure of O2 antigen.

FIGS. 20A and 20B: Analysis of LPS from clinical isolates with an O2positive agglutination phenotype. FIG. 20A. Silver staining. FIG. 20B.Western blotting using anti-O2 antiserum.

FIG. 21: OPS analysis from strain W3110 ΔwaaL ΔrfbW3110::rfbO2 ΔwekS. Achromatogram of 2AB labelled LLO analysis by normal phase HPLC is shown.

FIG. 22: Recognition of O25A and O25B LPS by anti-O25A and anti-O25B MBPantisera in a Western blotting analysis. Two nitrocellulose membraneswhich were obtained after electrotransfer of LPS samples prepared fromupec436 (O25A) and upec138 (O25B) and separated by a SDS-PAGE. Theloading pattern was identical for both membranes, left lane: O25A LPSfrom upec438, middle lane: O25B LPS from upec138. MBP bioconjugates wereused for immunization of rabbits. Left panel: anti O25B-MBP antiserum;right panel: anti O25A-MBP antiserum.

FIG. 23: MS/MS spectra of O2 OPS BRU. MS/MS spectrum of Na+ adduct withm/z=989.4 from elution peak at 43.5 min from 2AB labelled LLO extractsfrom strain CCUG25. The O2 BRU cartoon and the associated Y ion seriesis indicated confirming the expected monosaccharide sequence.

FIG. 24: EPA bioconjugates containing the O1A, O2, and O6 antigens usedin the preclinical study. OPS glycans were produced and purified, andanalyzed by SDS PAGE and visualized by Coomassie staining.

FIG. 25: shows mean ELISA titers obtained with sera from rats immunizedwith O1A-EPA (G1), carrier protein alone (G10), TBS (G11), or atetravalent composition composed of EPA-O1A, O2, O6Glc, and O25B (G12),probed against an ELISA plate coated with O1A-LPS purified from strainupec032.

FIG. 26: shows mean ELISA titers obtained with sera from rats immunizedwith O2-EPA (G4), carrier protein alone (G10), TBS (G11), or atetravalent composition composed of EPA-O1A, O2, O6Glc, and O25B (O12),probed against an ELISA plate coated with O2 LPS purified from strainsCCUG25.

FIG. 27: shows mean ELISA titers obtained with sera from rats immunizedwith O6Glc-EPA (G7), carrier protein alone (G10), TBS (G11), or atetravalent composition composed of EPA-O1A, O2, O6Glc, and O25B (G12),probed against an ELISA plate coated with O6Glc-LPS purified from strainCCUG11309.

FIG. 28: shows mean ELISA titers obtained with sera from rats immunizedwith O25B-EPA (G9), carrier protein alone (G10), TBS (G11), or atetravalent composition composed of O1A, O2, O6Glc, and O25B (O12),probed against an ELISA plate coated with O25B-LPS purified from strainupec177.

FIGS. 29A-29C: Opsonization indices of sera derived from ratspre-immunization (empty circles) compared to 42 days post-immunization(filled squares) with one priming dose and two booster doses ofindicated doses of monovalent vaccine. FIG. 29A shows O2-EPAimmunization; FIG. 29B shows O6-EPA immunization; FIG. 29C showsO25B-EPA immunization.

FIG. 30: ELISA titers obtained with sera from human subjects vaccinatedwith a tetravalent vaccine comprising E. coli antigens O1A, O2, O6Glc,and O25B. A significant increase in the ELISA titers between post (30days after injection) and pre-injection (day 1) was observed only in thevaccinated groups (*, represents statistical significance).

FIGS. 31A-31D: Opsonic Index (OI) obtained with sera from human subjectsvaccinated with a tetravalent vaccine comprising E. coli antigens O1A,O2, O6Glc, and O25B. Immune response as indicated by OI against placeboand components of the tetravalent vaccine (O1A-EPA (FIG. 31A), O2-EPA(FIG. 31B), O6Glc-EPA (FIG. 31C), and O25B-EPA (FIG. 31D)) before andafter injection are depicted. Pre-injection, defined as day 1, isrepresented by V2 (visit 2), and post-injection, defined as day 30, isrepresented by V4 (visit 4). A significant increase in the O1 betweenpost- and pre-injection (indicated by *, where multiple * representincreased degree of significance) was observed only in the vaccinatedgroups. NS, no significant difference.

FIG. 32: ELISA titers (expressed as EC50 values) of sera from vaccinatedsubjects toward O25A LPS (black bars) and O25B LPS (grey bars), at day 1(pre-vaccination) and after 30 days (post-vaccination). A statisticallysignificant increase in the ELISA titers between post-injection (30 daysafter injection) and pre-injection (day 1) was observed for bothserotypes: O25A LPS (black bars) and O25B LPS (grey bars).

FIG. 33: Reactivity of sera from vaccinated subjects toward O25A (blacklines) and O25B (grey lines) expressing E. coli strains. Dotted greyline: serotype O75 strain, a negative control. FIG. 33 demonstrates thatvaccine-induced serum IgG antibodies from vaccinated subjects stronglyrespond to O25A and O25B strains.

5. DETAILED DESCRIPTION

Disclosed herein are the structure of the E. coli antigen O25B, as wellas uses of O25B, methods of making of O25B, and bioconjugates comprisingof O25B. Applicants have identified the E. coli gene cluster responsiblefor production of O25B and have fully characterized the structure of theO25B antigen. Accordingly, provided herein are nucleic acids capable ofproducing O25B in host cells. Also provided herein are host cells, e.g.,recombinantly engineered host cells, comprising nucleic acids capable ofO25B production. Such host cells can be used to generate bioconjugatescomprising O25B linked to a carrier protein, which can be used in, e.g.,the formulation of therapeutics (e.g., vaccines). The O25B antigendescribed herein also is useful in the generation of antibodies, whichcan be used, e.g., in therapeutic methods such as passive immunizationof subjects. Further provided herein are compositions comprising O25B,alone or in combination with other E. coli antigens (e.g., O1, O2, andO6 and subserotypes thereof), for use in therapeutic methods, e.g.,vaccination of hosts against infection with E. coli (e.g., uropathogenicE. coli).

5.1 Nucleic Adds and Proteins

In one aspect, provided herein are isolated nucleic acids related toO25B production, e.g., nucleic acids encoding one or more proteins of anE. coli O25B rjb cluster. Those skilled in the art will appreciate thatdue to the degeneracy of the genetic code, a protein having a specificamino acid sequence can be encoded by multiple different nucleic acids.Thus, those skilled in the art will understand that a nucleic acidprovided herein can be altered in such a way that its sequence differsfrom a sequence provided herein, without affecting the amino acidsequence of the protein encoded by the nucleic acid.

In a specific embodiment, provided herein is a nucleic acid encoding anE. coli O25B rfb cluster. In a specific embodiment, provided herein is anucleic acid encoding an E. coli rfb(upec138) gene cluster (SEQ IDNO:12). In another specific embodiment, provided herein is a nucleicacid encoding a gene cluster that is about 750/, 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to or homologous to SEQ ID NO:12.Upec138 is an example of an E. coli strain of O25B serotype. The skilledperson will realize that other strains from this serotype can now easilybe obtained from clinical isolates according to methods describedherein, and examples of such other strains are upec177 and upec163.Hence, wherever an rfb gene cluster or individual genes from suchcluster of such O25B strains are mentioned herein, it is meant toinclude the corresponding gene clusters or genes from other O25Bstrains. Also the sequences provided can be found by sequencing the rjbgene clusters or if desired of individual genes from such otherisolates, and will provide homologous sequences encoding homologousproteins as the gene cluster or gene. In any embodiments where ahomologous gene cluster or gene is mentioned by referring to a genecluster or gene with a certain percentage, such homologous sequencepreferably encodes the protein(s) with the same function as the onesfrom the reference strain or sequence.

In another specific embodiment, provided herein is a nucleic acidencoding an E. coli O25B rfb cluster. In a specific embodiment, providedherein is a nucleic acid encoding an E. coli rjb(upec163) gene cluster.In another specific embodiment, provided herein is a nucleic acidencoding a gene cluster that is about 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to or homologous to an E. coli rjb(upec163) genecluster.

In another specific embodiment, provided herein is a nucleic acidencoding an E. coli O25B rfb cluster. In a specific embodiment, providedherein is a nucleic acid encoding an E. coli rfb(upec177) gene cluster.In another specific embodiment, provided herein is a nucleic acidencoding a gene cluster that is about 750/, 800/, 85%, 90%, 95%, 96%,97%, 98%, or 99% identical to or homologous to an E. coli rfb(upec177)gene cluster.

In another embodiment, provided herein are nucleic acid encodingproteins of an E. coli O25B rfb cluster.

In a specific embodiment, a nucleic acid encoding a protein of an E.coli O25B rib cluster provided herein comprises or consists of SEQ IDNO: 1, the rmlB gene of the E. coli O25B rfb cluster. In anotherspecific embodiment, a nucleic acid encoding a protein of an E. coliO25B rfb cluster provided herein is 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to or homologous to SEQ ID NO:1.

In a specific embodiment, a nucleic acid encoding a protein of an E.coli O25B rib cluster provided herein comprises or consists of SEQ IDNO:2, the rmlD gene of the E. coli O25B rfb cluster. In another specificembodiment, a nucleic acid encoding a protein of an E. coli O25B rfbcluster provided herein is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% identical to or homologous to SEQ ID NO:2.

In a specific embodiment, a nucleic acid encoding a protein of an E.coli O25B rjb cluster provided herein comprises or consists of SEQ IDNO:3, the rmlA gene of the E. coli O25B rfb cluster. In another specificembodiment, a nucleic acid encoding a protein of an E. coli O25B rfbcluster provided herein is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% identical to or homologous to SEQ ID NO:3.

In a specific embodiment, a nucleic acid encoding a protein of an E.coli O25B rib cluster provided herein comprises or consists of SEQ IDNO:4, the rmlC gene of the E. coli O25B rfb cluster. In another specificembodiment, a nucleic acid encoding a protein of an E. coli O25B rfbcluster provided herein is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% identical to or homologous to SEQ ID NO:4.

In a specific embodiment, a nucleic acid encoding a protein of an E.coli O25B rjb cluster provided herein comprises or consists of SEQ IDNO:5, the wzx gene of the E. coli O25B rfb cluster. In another specificembodiment, a nucleic acid encoding a protein of an E. coli O25B rfbcluster provided herein is 75%, 80%, 85%, 90%0, 95%, 96%, 97%, 98%, or99% identical to or homologous to SEQ ID NO:5.

In a specific embodiment, a nucleic acid encoding a protein of an E.coli O25B rb cluster provided herein comprises or consists of SEQ IDNO:6, the wekA gene of the E. coli O25B rjb cluster. In another specificembodiment, a nucleic acid encoding a protein of an E. coli O25B rfbcluster provided herein is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% identical to or homologous to SEQ ID NO:6.

In a specific embodiment, a nucleic acid encoding a protein of an E.coli O25B rib cluster provided herein comprises or consists of SEQ IDNO:7, the wekB gene of the E. coli O25B rfb cluster. In another specificembodiment, a nucleic acid encoding a protein of an E. coli O25B rfbcluster provided herein is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% identical to or homologous to SEQ ID NO:7.

In a specific embodiment, a nucleic acid encoding a protein of an E.coli O25B rib cluster provided herein comprises or consists of SEQ IDNO:8, the wzy gene of the E. coli O25B rfb cluster. In another specificembodiment, a nucleic acid encoding a protein of an E. coli O25B ribcluster provided herein is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% identical to or homologous to SEQ ID NO:8.

In a specific embodiment, a nucleic acid encoding a protein of an E.coli O25B rib cluster provided herein comprises or consists of SEQ IDNO:9, the wbbJ gene of the E. coli zO25B rfb cluster. In anotherspecific embodiment, a nucleic acid encoding a protein of an E. coliO25B rfb cluster provided herein is 75%, 80%, 85%, 90%, 95%, 96%, 97%Y/, 98%, or 99% identical to or homologous to SEQ ID NO:9.

In a specific embodiment, a nucleic acid encoding a protein of an E.coli O25B rfb cluster provided herein comprises or consists of SEQ IDNO:10, the wbbK gene of the E. coli O25B rfb cluster. In anotherspecific embodiment, a nucleic acid encoding a protein of an E. coliO25B rjb cluster provided herein is 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to or homologous to SEQ ID NO:10.

In a specific embodiment, a nucleic acid encoding a protein of an E.coli O25B rib cluster provided herein comprises or consists of SEQ IDNO: 11, the wbbL gene of the E. coli O25B rb cluster. In anotherspecific embodiment, a nucleic acid encoding a protein of an E. coliO25B rfb cluster provided herein is 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to or homologous to SEQ ID NO: 11.

In another aspect, provided herein are proteins encoded by the nucleicacids provided herein. In a specific embodiment, provided herein isdTDP-Glucose 4,6-dehydratase, encoded by SEQ ID NO:1. In anotherspecific embodiment, provided herein is dTDP-6-Deoxy-D-glucose3,5-epimerase, encoded by SEQ ID NO:2. In another specific embodiment,provided herein is Glucose-1-phosphate thymidylyltransferase, encoded bySEQ ID NO:3. In another specific embodiment, provided herein isdTDP-4-dehydrorhamnose 3,5-epimerase, encoded by SEQ ID NO:4. In anotherspecific embodiment, provided herein is O antigen flippase, encoded bySEQ ID NO:5. In another specific embodiment, provided herein isdTDP-Rha:Glc-Rha(Ac)-GlcNAc-UPP α-1,3-rhamnosyltransferase, encoded bySEQ ID NO:6. In another specific embodiment, provided herein isUDP-Glc:Glc-Rha(Ac)-GlcNAc-UPP fl-1,6-glucosyltransferase, encoded bySEQ ID NO:7. In another specific embodiment, provided herein is Oantigen polymerase, encoded by SEQ ID NO:8. In another specificembodiment, provided herein is O-acetyl transferase, encoded by SEQ IDNO:9. In another specific embodiment, provided herein isUDP-Glc:Rha-GlcNAc-UPP α-1,3-glucosyltransferase, encoded by SEQ IDNO:10. In another specific embodiment, provided herein isdTDP-Rha:GlcNAc-UPP α-1,3-rhamnosyltransferase, encoded by SEQ ID NO:11.

5.2 E. coli O Antigens

In one aspect, provided herein are isolated E. coli antigens of the O25,O1, O2, and O6 serotypes.

In a specific embodiment, provided herein is an isolated O antigen fromE. coli strain upec138. In another specific embodiment, provided hereinis an isolated O antigen from E. coli strain upec163. In anotherspecific embodiment, provided herein is an isolated O antigen from E.coli strain upec177.

In another specific embodiment, provided herein is an isolated E. coliO25B antigen of Formula O25B:

In another specific embodiment, provided herein is an isolated E. coliO25B antigen of Formula O25B′:

In another aspect, provided herein is a population of isolatedmacromolecules of the Formula O25B, presented below:

wherein n is an integer between 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25,20 to 25, 10 to 20, or 15 to 20. In a specific embodiment, n of at least80% of the macromolecules in the population is between 1 to 30, 1 to 20,1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20.

In another aspect, provided herein a population of isolatedmacromolecules of the Formula O25B′, presented below:

wherein n is an integer between 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25,20 to 25, 10 to 20, or 15 to 20. In a specific embodiment, n of at least80% of the macromolecules in the population is between 1 to 30, 1 to 20,1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20.

Other E. coli antigens useful in the compositions described herein(e.g., therapeutic compositions, e.g., vaccines; see Section 5.6)include O25A, as well as O1, O2, and O6 antigens, and subserotypesthereof.

In one embodiment, an O25A antigen (e.g., in isolated form or as part ofa bioconjugate) is used in a composition provided herein (e.g., incombination with an O25B antigen (or bioconjugate comprising an O25Bantigen)). In a specific embodiment, the O25A antigen is Formula O25A:

In another specific embodiment, the O25A antigen is Formula O25A′:

In one embodiment, an O1A antigen (e.g., in isolated form or as part ofa bioconjugate) is used in a composition provided herein (e.g., incombination with an O25B antigen (or bioconjugate comprising an O25Bantigen)). In a specific embodiment, the O1A antigen is Formula O1A:

In another specific embodiment, the O A antigen is Formula O1A′:

In one embodiment, an O1B antigen (e.g., in isolated form or as part ofa bioconjugate) is used in a composition provided herein (e.g., incombination with an O25B antigen (or bioconjugate comprising an O25Bantigen)). In a specific embodiment, the O1B antigen is Formula O1B:

In another specific embodiment, the O1B antigen is Formula O1B′:

In one embodiment, an OIC antigen (e.g., in isolated form or as part ofa bioconjugate) is used in a composition provided herein (e.g., incombination with an O25B antigen (or bioconjugate comprising an O25Bantigen)). In a specific embodiment, the O1C antigen is Formula O1C:

In another specific embodiment, the O1C antigen is Formula O1C′:

In one embodiment, an O2 antigen (e.g., in isolated form or as part of abioconjugate) is used in a composition provided herein (e.g., incombination with an O25B antigen (or bioconjugate comprising an O25Bantigen)). In a specific embodiment, the O2 antigen is Formula O2:

In another specific embodiment, the O2 antigen is Formula O2′:

In one embodiment, an O6 antigen (e.g., in isolated form or as part of abioconjugate) is used in a composition provided herein (e.g., incombination with an O25B antigen (or bioconjugate comprising an O25Bantigen)). In a specific embodiment, the O6 antigen is Formula O6K2(also referred to herein as O6Glc):

In another specific embodiment, the O6 antigen is Formula O6K2′ (alsoreferred to herein as O6Glc′):

In another specific embodiment, the O6 antigen is Formula O6K54 (alsoreferred to herein as O6GlcNAc):

In another specific embodiment, the O6 antigen is Formula O6K54′ (alsoreferred to herein as O6GlcNAc′):

5.3 Host Cells

Provided herein are host cells, e.g., prokaryotic host cells, capable ofproducing E. coli O antigens and bioconjugates comprising such E. coli Oantigens. In certain embodiments, the host cells provided hereincomprise (e.g., naturally or through genetic engineering) one or more ofthe nucleic acids described herein. See Section 5.1. In certainembodiments, the host cells provided herein produce one or more of theE. coli O antigens described herein, and/or produce bioconjugatescomprising one or more of the E. coli O antigens described herein. SeeSection 5.2.

In one aspect, provided herein is a prokaryotic host cell comprisingnucleic acids encoding enzymes (e.g., glycosyltransferases) capable ofproducing the novel polysaccharide disclosed herein, E. coli O25B. Alsoprovided herein are host cells comprising nucleic acids encoding enzymes(e.g., glycosyltransferases) capable of producing other E. coliantigens, e.g., O25A, O1, O2, and O6, and subserotypes thereof (seeSection 5.2). The host cells provided herein may naturally expressnucleic acids capable of producing of an O antigen of interest, or thehost cells may be made to express such nucleic acids, i.e., in certainembodiments said nucleic acids are heterologous to the host cells andintroduced into the host cells using genetic approaches known in theart. In certain embodiments, the host cells provided herein comprisenucleic acids encoding additional enzymes active in the N-glycosylationof proteins, e.g., the host cell provided herein can further comprise anucleic acid encoding an oligosaccharyl transferase or nucleic acidsencoding other glycosyltransferases. See, e.g., Section 5.3.3. Incertain embodiments, the host cells provided herein comprise a nucleicacid encoding a carrier protein, e.g., a protein to which oligo- andpolysaccharides can be attached to form a bioconjugate. See, e.g.,Section 5.3.2 for a description of carrier proteins and Section 5.4 fora description of bioconjugates. In a specific embodiment, the host cellis E. coli.

Upec138 is an E. coli strain identified herein as belonging to the O25Bserotype, and the rfb gene cluster of the strain (and strains of theO25B serotype in general) has been identified herein for the first timeas comprising genes that produce a novel E. coli polysaccharide, O25B.In a specific embodiment, provided herein is a prokaryotic host cellcomprising an E. coli rfb(upec138) gene cluster (SEQ ID NO:12), or agene cluster that is about or at least 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identical to or homologous to SEQ ID NO: 12. In aspecific embodiment, the E. coli rfb(upec138) gene cluster (SEQ IDNO:12) is introduced into the host cell by genetic manipulation (e.g.,the gene cluster is expressed on a plasmid or plasmids or integratedinto the host cell genome (see, e.g., International Patent applicationNo. PCT/EP2013/068737)). In another specific embodiment, the prokaryotichost cell comprises a nucleic acid sequence encoding an oligosaccharyltransferase. In another specific embodiment, the prokaryotic host cellfurther comprises a nucleic acid sequence encoding a carrier proteincomprising a consensus sequence Asn-X-Ser(Thr), wherein X can be anyamino acid except Pro (SEQ ID NO:14) or a carrier protein comprising aconsensus sequence or Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z areindependently selected from any natural amino acid except Pro (SEQ IDNO:15) (see WO 2006/119987). In another specific embodiment, some or allof the genes of the rfb cluster are heterologous to the host cell. Inanother specific embodiment, said oligosaccharyl transferase isheterologous to the host cell. In another specific embodiment, saidcarrier protein is heterologous to the host cell. In a specificembodiment, the host cell is E. coli.

Upec163 is an E. coli strain identified herein as belonging to the O25Bserotype, and the rfb gene cluster of the strain (and strains of theO25B serotype in general) has been identified herein for the first timeas comprising genes that produce a novel E. coli polysaccharide, O25B.In another specific embodiment, provided herein is a prokaryotic hostcell comprising an E. coli rfb(upec163) gene cluster, or a gene clusterthat is about or at least 75%, 80%, 85%, 90%, 95/%, 96%, 97%, 98%, or99% identical to or homologous to an E. coli rfb(upec163) gene cluster.In a specific embodiment, the E. coli rtb(upec163) gene cluster isintroduced into the host cell by genetic manipulation (e.g., the genecluster is expressed on a plasmid or plasmids or integrated into thehost cell genome (see, e.g., International Patent application No.PCT/EP2013/068737)). In another embodiment, the prokaryotic host cellcomprises a nucleic acid sequence encoding an oligosaccharyltransferase. In another specific embodiment, the prokaryotic host cellfurther comprises a nucleic acid sequence encoding a carrier proteincomprising a consensus sequence Asn-X-Ser(Thr), wherein X can be anyamino acid except Pro (SEQ ID NO:14) or a carrier protein comprising aconsensus sequence or Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z areindependently selected from any natural amino acid except Pro (SEQ IDNO:15) (see WO 2006/119987). In another specific embodiment, some or allof the genes of the rfb cluster are heterologous to the host cell. Inanother specific embodiment, said oligosaccharyl transferase isheterologous to the host cell. In another specific embodiment, saidcarrier protein is heterologous to the host cell. In a specificembodiment, the host cell is E. coli.

Upec177 is an E. coli strain identified herein as belonging to the O25Bserotype, and the rfb gene cluster of the strain (and strains ofthe O25Bserotype in general) has been identified herein for the first time ascomprising genes that produce a novel E. coli polysaccharide, O25B. Inanother specific embodiment, provided herein is a prokaryotic host cellcomprising an E. coli rfb(upec177) gene cluster, or a gene cluster thatis about or at least 75%, 80%, 85%, 900/, 95%, 96%, 97%, 98%, or 99%identical to or homologous to an E. coli rfb(upec177) gene cluster. In aspecific embodiment, the E. coli rfb(upec177) gene cluster is introducedinto the host cell by genetic manipulation (e.g., the gene cluster isexpressed on a plasmid or plasmids or integrated into the host cellgenome (see, e.g., International Patent application No.PCT/EP2013/068737)). In another embodiment, the prokaryotic host cellcomprises a nucleic acid sequence encoding an oligosaccharyltransferase. In another specific embodiment, the prokaryotic host cellfurther comprises a nucleic acid sequence encoding a carrier proteincomprising a consensus sequence Asn-X-Ser(Thr), wherein X can be anyamino acid except Pro (SEQ ID NO:14) or a carrier protein comprising aconsensus sequence or Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z areindependently selected from any natural amino acid except Pro (SEQ IDNO: 15) (see WO 2006/119987). In another specific embodiment, some orall of the genes of the rfb cluster are heterologous to the host cell.In another specific embodiment, said oligosaccharyl transferase isheterologous to the host cell. In another specific embodiment, saidcarrier protein is heterologous to the host cell. In a specificembodiment, the host cell is E. coli.

In another specific embodiment, provided herein is a prokaryotic hostcell (e.g., a recombinantly engineered prokaryotic host cell) thatproduces O25B, wherein said host cell comprises rmlB, rmlD, rmlA, rmlC,wzr, wekA, wekB, wzy, wbbJ, wbbK, and/or wbbL. Such host cells can beengineered using recombinant approaches to comprise one or more plasmidscomprising the rmlB, rmlD, rmlA, rmlC, wzx, wekA, wekB, wzy, wbbJ, wbbK,and/or wbbL genes. In certain embodiments, said one or more plasmids isintegrated into the host cell genome. In a specific embodiment, saidrmlB comprises or consists of SEQ ID NO: 1, or is 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% identical to or homologous to SEQ ID NO: 1.In a specific embodiment, said rmlD comprises or consists of SEQ IDNO:2, or is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical toor homologous to SEQ ID NO:2. In a specific embodiment, said rmlAcomprises or consists of SEQ ID NO:3, or is 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to or homologous to SEQ ID NO:3. In aspecific embodiment, said rmlC comprises or consists of SEQ ID NO:4, oris 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to orhomologous to SEQ ID NO:4. In a specific embodiment, said wzx comprisesor consists of SEQ ID NO:5, or is 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to or homologous to SEQ ID NO:5. In a specificembodiment, said wekA comprises or consists of SEQ ID NO:6, or is 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or homologous toSEQ ID NO:6. In a specific embodiment, said wekB comprises or consistsof SEQ ID NO:7, or is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to or homologous to SEQ ID NO:7. In a specific embodiment,said wzy comprises or consists of SEQ ID NO:8, or is 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% identical to or homologous to SEQ ID NO:8. Ina specific embodiment, said wbbJ comprises or consists of SEQ ID NO:9,or is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to orhomologous to SEQ ID NO:9. In a specific embodiment, said wbbK comprisesor consists of SEQ ID NO:10, or is 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to or homologous to SEQ ID NO: 10. In a specificembodiment, said wbbL comprises or consists of SEQ ID NO: 11, or is 75%,80%, 85%, 900/, 95%, 96%, 97%, 98%, or 99% identical to or homologous toSEQ ID NO: 11. In another specific embodiment, the prokaryotic host cellcomprises a nucleic acid sequence encoding an oligosaccharyltransferase. In another specific embodiment, the prokaryotic host cellcomprises a nucleic acid sequence encoding a carrier protein comprisinga consensus sequence Asn-X-Ser(Thr), wherein X can be any amino acidexcept Pro (SEQ ID NO:14) or a carrier protein comprising a consensussequence or Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independentlyselected from any natural amino acid except Pro (SEQ ID NO:15) (see WO2006/119987). In another specific embodiment, some or all of the genes,rmlB, rmlD, rmlA, rmlC, warx, wekA, wekB, wzy, wbbJ, wbbK, and wbbL, areheterologous to the host cell. In another specific embodiment, saidoligosaccharyl transferase is heterologous to the host cell. In anotherspecific embodiment, said carrier protein is heterologous to the hostcell. In a specific embodiment, the host cell is E. coli.

In another specific embodiment, provided herein is a prokaryotic hostcell (e.g., a recombinantly engineered prokaryotic host cell) thatproduces O25B, wherein said host cell comprises one, two, three, four,or more, e.g. all, of the following genes (or a nucleic acid that isabout or at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to or homologous to one of the following genes, and preferablyencoding protein with the same function): rmlB (SEQ ID NO:1), rmlD (SEQID NO:2), rmlA (SEQ ID NO:3), rmlC (SEQ ID NO:4), wzx (SEQ ID NO:5),wekA (SEQ ID NO:6), wekB (SEQ ID NO:7), wzy (SEQ ID NO:8), wbbJ (SEQ IDNO:9), wbbK (SEQ ID NO: 10), and/or wbbL (SEQ ID NO: 11). In anotherspecific embodiment, the prokaryotic host cell comprises a nucleic acidsequence encoding an oligosaccharyl transferase. In another specificembodiment, the prokaryotic host cell further comprises a nucleic acidsequence encoding a carrier protein comprising a consensus sequenceAsn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ IDNO:14); or a carrier protein comprising a consensus sequenceAsp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selectedfrom any natural amino acid except Pro (SEQ ID NO: 15) (see WO2006/119987). In a specific embodiment, the host cell is E. coli.

In another specific embodiment, provided herein is a prokaryotic hostcell (e.g., a recombinantly engineered prokaryotic host cell) capable ofproducing O25B, and/or an O25B bioconjugate (i.e., a carrier proteinlinked to the E. coli O25B antigen), wherein said host cell naturallycomprises or is engineered to comprise the nucleic acid sequence of rmlB(SEQ ID NO:1). In another specific embodiment, provided herein is aprokaryotic host cell capable of producing O25B, and/or an O25Bbioconjugate (i.e., a carrier protein linked to the E. coli O25Bantigen), wherein said host cell naturally comprises or is engineered tocomprise a nucleic sequence that encodes a dTDP-Glucose 4,6-dehydratase,e.g., a dTDP-Glucose 4,6-dehydratase encoded by rmlB. In anotherspecific embodiment, provided herein is a prokaryotic host cell capableof producing O25B, and/or an O25B bioconjugate (i.e., a carrier proteinlinked to the E. coli O25B antigen), wherein said host cell naturallycomprises or is engineered to comprise a nucleic acid that is about orat least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 890/, 900/, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ IDNO:1. In another specific embodiment, the prokaryotic host cellcomprises a nucleic acid sequence encoding an oligosaccharyltransferase. In another specific embodiment, the prokaryotic host cellfurther comprises a nucleic acid sequence encoding a carrier proteincomprising a consensus sequence Asn-X-Ser(Thr), wherein X can be anyamino acid except Pro (SEQ ID NO:14); or a carrier protein comprising aconsensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z areindependently selected from any natural amino acid except Pro (SEQ IDNO: 15) (see WO 2006/119987). In a specific embodiment, the host cell isE. coli.

In another specific embodiment, provided herein is a prokaryotic hostcell (e.g., a recombinantly engineered prokaryotic host cell) capable ofproducing O25B, and/or an O25B bioconjugate (i.e., a carrier proteinlinked to the E. coli O25B antigen), wherein said host cell naturallycomprises or is engineered to comprise the nucleic acid sequence of rmlD(SEQ ID NO:2). In another specific embodiment, provided herein is aprokaryotic host cell capable of producing O25B, and/or an O25Bbioconjugate (i.e., a carrier protein linked to the E. coli O25Bantigen), wherein said host cell naturally comprises or is engineered tocomprise a nucleic sequence that encodes a dTDP-6-Deoxy-D-glucose3,5-epimerase, e.g., a dTDP-6-Deoxy-D-glucose 3,5-epimerase encoded byrmlD. In another specific embodiment, provided herein is a prokaryotichost cell capable of producing O25B, and/or an O25B bioconjugate (i.e.,a carrier protein linked to the E. coli O25B antigen), wherein said hostcell naturally comprises or is engineered to comprise a nucleic acidthat is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ IDNO:2. In another specific embodiment, the prokaryotic host cellcomprises a nucleic acid sequence encoding an oligosaccharyltransferase. In another specific embodiment, the prokaryotic host cellfurther comprises a nucleic acid sequence encoding a carrier proteincomprising a consensus sequence Asn-X-Ser(Thr), wherein X can be anyamino acid except Pro (SEQ ID NO:14); or a carrier protein comprising aconsensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z areindependently selected from any natural amino acid except Pro (SEQ IDNO:15) (see WO 2006/119987). In a specific embodiment, the host cell isE. coli.

In another specific embodiment, provided herein is a prokaryotic hostcell (e.g., a recombinantly engineered prokaryotic host cell) capable ofproducing O25B, and/or an O25B bioconjugate (i.e., a carrier proteinlinked to the E. coli O25B antigen), wherein said host cell naturallycomprises or is engineered to comprise the nucleic acid sequence of rmlA(SEQ ID NO:3). In another specific embodiment, provided herein is aprokaryotic host cell capable of producing O25B, and/or an O25Bbioconjugate (i.e., a carrier protein linked to the E. coli O25Bantigen), wherein said host cell naturally comprises or is engineered tocomprise a nucleic sequence that encodes a Glucose-1-phosphatethymidylyltransferase, e.g., a Glucose-1-phosphate thymidylyltransferaseencoded by rmlA. In another specific embodiment, provided herein is aprokaryotic host cell capable of producing O25B, and/or an O25Bbioconjugate (i.e., a carrier protein linked to the E. coli O25Bantigen), wherein said host cell naturally comprises or is engineered tocomprise a nucleic acid that is at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 980/, 99%,or 100% identical to SEQ ID NO:3. In another specific embodiment, theprokaryotic host cell comprises a nucleic acid sequence encoding anoligosaccharyl transferase. In another specific embodiment, theprokaryotic host cell further comprises a nucleic acid sequence encodinga carrier protein comprising a consensus sequence Asn-X-Ser(Thr),wherein X can be any amino acid except Pro (SEQ ID NO:14); or a carrierprotein comprising a consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr),wherein X and Z are independently selected from any natural amino acidexcept Pro (SEQ ID NO:15) (see WO 2006/119987). In a specificembodiment, the host cell is E. coli.

In another specific embodiment, provided herein is a prokaryotic hostcell (e.g., a recombinantly engineered prokaryotic host cell) capable ofproducing O25B, and/or an O25B bioconjugate (i.e., a carrier proteinlinked to the E. coli O25B antigen), wherein said host cell naturallycomprises or is engineered to comprise the nucleic acid sequence of rmlC(SEQ ID NO:4). In another specific embodiment, provided herein is aprokaryotic host cell capable of producing O25B, and/or an O25Bbioconjugate (i.e., a carrier protein linked to the E. coli O25Bantigen), wherein said host cell naturally comprises or is engineered tocomprise a nucleic sequence that encodes a dTDP-4-dehydrorhamnose3,5-epimerase, e.g., a dTDP-4-dehydrorhamnose 3,5-epimerase encoded byrmlC. In another specific embodiment, provided herein is a prokaryotichost cell capable of producing O25B, and/or an O25B bioconjugate (i.e.,a carrier protein linked to the E. coli O25B antigen), wherein said hostcell naturally comprises or is engineered to comprise a nucleic acidthat is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ IDNO:4. In another specific embodiment, the prokaryotic host cellcomprises a nucleic acid sequence encoding an oligosaccharyltransferase. In another specific embodiment, the prokaryotic host cellfurther comprises a nucleic acid sequence encoding a carrier proteincomprising a consensus sequence Asn-X-Ser(Thr), wherein X can be anyamino acid except Pro (SEQ ID NO:14); or a carrier protein comprising aconsensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z areindependently selected from any natural amino acid except Pro (SEQ IDNO:15) (see WO 2006/119987). In a specific embodiment, the host cell isE. coli.

In another specific embodiment, provided herein is a prokaryotic hostcell (e.g., a recombinantly engineered prokaryotic host cell) capable ofproducing O25B, and/or an O25B bioconjugate (i.e., a carrier proteinlinked to the E. coli O25B antigen), wherein said host cell naturallycomprises or is engineered to comprise the nucleic acid sequence of wzx(SEQ ID NO:5). In another specific embodiment, provided herein is aprokaryotic host cell capable of producing O25B, and/or an O25Bbioconjugate (i.e., a carrier protein linked to the E. coli O25Bantigen), wherein said host cell naturally comprises or is engineered tocomprise a nucleic sequence that encodes an O antigen flippase, e.g., anO antigen flippase encoded by wzx. In another specific embodiment,provided herein is a prokaryotic host cell capable of producing O25B,and/or an O25B bioconjugate (i.e., a carrier protein linked to the E.coli O25B antigen), wherein said host cell naturally comprises or isengineered to comprise a nucleic acid that is at least 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to SEQ ID NO:5. In another specificembodiment, the prokaryotic host cell comprises a nucleic acid sequenceencoding an oligosaccharyl transferase. In another specific embodiment,the prokaryotic host cell further comprises a nucleic acid sequenceencoding a carrier protein comprising a consensus sequenceAsn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ IDNO:14); or a carrier protein comprising a consensus sequenceAsp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selectedfrom any natural amino acid except Pro (SEQ ID NO:15) (see WO2006/119987). In a specific embodiment, the host cell is E. coli.

In another specific embodiment, provided herein is a prokaryotic hostcell (e.g., a recombinantly engineered prokaryotic host cell) capable ofproducing O25B, and/or an O25B bioconjugate (i.e., a carrier proteinlinked to the E. coli O25B antigen), wherein said host cell naturallycomprises or is engineered to comprise the nucleic acid sequence of wekA(SEQ ID NO:6). In another specific embodiment, provided herein is aprokaryotic host cell capable of producing O25B, and/or an O25Bbioconjugate (i.e., a carrier protein linked to the E. coli O25Bantigen), wherein said host cell naturally comprises or is engineered tocomprise a nucleic sequence that encodes a rhamnosyltransferase, e.g.,an dTDP-Rha:Glc-Rha(Ac)-GlcNAc-UPP α-1,3-rhamnosyltransferase encoded bywekA. In another specific embodiment, provided herein is a prokaryotichost cell capable of producing O25B, and/or an O25B bioconjugate (i.e.,a carrier protein linked to the E. coli O25B antigen), wherein said hostcell naturally comprises or is engineered to comprise nucleic acids thatis at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ IDNO:6. In another specific embodiment, the prokaryotic host cellcomprises a nucleic acid sequence encoding an oligosaccharyltransferase. In another specific embodiment, the prokaryotic host cellfurther comprises a nucleic acid sequence encoding a carrier proteincomprising a consensus sequence Asn-X-Ser(Thr), wherein X can be anyamino acid except Pro (SEQ ID NO:14); or a carrier protein comprising aconsensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z areindependently selected from any natural amino acid except Pro (SEQ IDNO: 15) (see WO 2006/119987). In a specific embodiment, the host cell isE. coli.

In another specific embodiment, provided herein is a prokaryotic hostcell (e.g., a recombinantly engineered prokaryotic host cell) capable ofproducing O25B, and/or an O25B bioconjugate (i.e., a carrier proteinlinked to the E. coli O25B antigen), wherein said host cell naturallycomprises or is engineered to comprise the nucleic acid sequence of wekB(SEQ ID NO:7). In another specific embodiment, provided herein is aprokaryotic host cell capable of producing O25B, and/or an O25Bbioconjugate (i.e., a carrier protein linked to the E. coli O25Bantigen), wherein said host cell naturally comprises or is engineered tocomprise a nucleic sequence that encodes a wekB glucosyltransferase,e.g., a UDP-Glc:Glc-Rha(Ac)-GlcNAc-UPP 1-1,6-glucosyltransferase encodedby wekB. In another specific embodiment, provided herein is aprokaryotic host cell capable of producing O25B, and/or an O25Bbioconjugate (i.e., a carrier protein linked to the E. coli O25Bantigen), wherein said host cell naturally comprises or is engineered tocomprise nucleic acids that is at least 80%, 81%, 82%, 83%, 84%, 85%,860%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97/%, 98%, 99%,or 100% identical to SEQ ID NO:7. In another specific embodiment, theprokaryotic host cell comprises a nucleic acid sequence encoding anoligosaccharyl transferase. In another specific embodiment, theprokaryotic host cell further comprises a nucleic acid sequence encodinga carrier protein comprising a consensus sequence Asn-X-Ser(Thr),wherein X can be any amino acid except Pro (SEQ ID NO:14); or a carrierprotein comprising a consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr),wherein X and Z are independently selected from any natural amino acidexcept Pro (SEQ ID NO:15) (see WO 2006/119987). In a specificembodiment, the host cell is E. coli.

In another specific embodiment, provided herein is a prokaryotic hostcell (e.g., a recombinantly engineered prokaryotic host cell) capable ofproducing O25B, and/or an O25B bioconjugate (i.e., a carrier proteinlinked to the E. coli O25B antigen), wherein said host cell naturallycomprises or is engineered to comprise the nucleic acid sequence of wzy(SEQ ID NO:8). In another specific embodiment, provided herein is aprokaryotic host cell capable of producing O25B, and/or an O25Bbioconjugate (i.e., a carrier protein linked to the E. coli O25Bantigen), wherein said host cell naturally comprises or is engineered tocomprise a nucleic sequence that encodes an O antigen polymerase, e.g.,an O antigen polymerase encoded by wzy.

In another specific embodiment, provided herein is a prokaryotic hostcell capable of producing O25B, and/or an O25B bioconjugate (i.e., acarrier protein linked to the E. coli O25B antigen), wherein said hostcell naturally comprises or is engineered to comprise nucleic acids thatis at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ IDNO:8. In another specific embodiment, the prokaryotic host cellcomprises a nucleic acid sequence encoding an oligosaccharyltransferase. In another specific embodiment, the prokaryotic host cellfurther comprises a nucleic acid sequence encoding a carrier proteincomprising a consensus sequence Asn-X-Ser(Thr), wherein X can be anyamino acid except Pro (SEQ ID NO:14); or a carrier protein comprising aconsensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z areindependently selected from any natural amino acid except Pro (SEQ IDNO: 15) (see WO 2006/119987). In a specific embodiment, the host cell isE. coli.

In another specific embodiment, provided herein is a prokaryotic hostcell (e.g., a recombinantly engineered prokaryotic host cell) capable ofproducing O25B, and/or an O25B bioconjugate (i.e., a carrier proteinlinked to the E. coli O25B antigen), wherein said host cell naturallycomprises or is engineered to comprise the nucleic acid sequence of wbbJ(SEQ ID NO:9). In another specific embodiment, provided herein is aprokaryotic host cell capable of producing O25B, and/or an O25Bbioconjugate (i.e., a carrier protein linked to the E. coli O25Bantigen), wherein said host cell naturally comprises or is engineered tocomprise a nucleic sequence that encodes an O-acetyl transferase, e.g.,an O-acetyl transferase encoded by wbbJ. In another specific embodiment,provided herein is a prokaryotic host cell capable of producing O25B,and/or an O25B bioconjugate (i.e., a carrier protein linked to the E.coli O25B antigen), wherein said host cell naturally comprises or isengineered to comprise nucleic acids that is at least 80%, 81%, 82/,83%, 84%, 85%, 86%, 87%, 88%/, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to SEQ ID NO:9. In another specificembodiment, the prokaryotic host cell comprises a nucleic acid sequenceencoding an oligosaccharyl transferase. In another specific embodiment,the prokaryotic host cell further comprises a nucleic acid sequenceencoding a carrier protein comprising a consensus sequenceAsn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ IDNO:14); or a carrier protein comprising a consensus sequenceAsp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selectedfrom any natural amino acid except Pro (SEQ ID NO: 15) (see WO2006/119987). In a specific embodiment, the host cell is E. coli.

In another specific embodiment, provided herein is a prokaryotic hostcell (e.g., a recombinantly engineered prokaryotic host cell) capable ofproducing O25B, and/or an O25B bioconjugate (i.e., a carrier proteinlinked to the E. coli O25B antigen), wherein said host cell naturallycomprises or is engineered to comprise the nucleic acid sequence of wbbK(SEQ ID NO: 10). In another specific embodiment, provided herein is aprokaryotic host cell capable of producing O25B, and/or an O25Bbioconjugate (i.e., a carrier protein linked to the E. coli O25Bantigen), wherein said host cell naturally comprises or is engineered tocomprise a nucleic sequence that encodes a glucosyltransferase, e.g., aUDP-Glc:Rha-GlcNAc-UPP α-1,3-glucosyltransferase encoded by wbbK. Inanother specific embodiment, provided herein is a prokaryotic host cellcapable of producing O25B, and/or an O25B bioconjugate (i.e., a carrierprotein linked to the E. coli O25B antigen), wherein said host cellnaturally comprises or is engineered to comprise nucleic acids that isat least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:10. In another specific embodiment, the prokaryotic host cell comprisesa nucleic acid sequence encoding an oligosaccharyl transferase. Inanother specific embodiment, the prokaryotic host cell further comprisesa nucleic acid sequence encoding a carrier protein comprising aconsensus sequence Asn-X-Ser(Thr), wherein X can be any amino acidexcept Pro (SEQ ID NO:14); or a carrier protein comprising a consensussequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independentlyselected from any natural amino acid except Pro (SEQ ID NO:15) (see WO2006/119987). In a specific embodiment, the host cell is E. coli.

In another specific embodiment, provided herein is a prokaryotic hostcell (e.g., a recombinantly engineered prokaryotic host cell) capable ofproducing O25B, and/or an O25B bioconjugate (i.e., a carrier proteinlinked to the E. coli O25B antigen), wherein said host cell naturallycomprises or is engineered to comprise the nucleic acid sequence of wbbL(SEQ ID NO:11). In another specific embodiment, provided herein is aprokaryotic host cell capable of producing O25B, and/or an O25Bbioconjugate (i.e., a carrier protein linked to the E. coli O25Bantigen), wherein said host cell naturally comprises or is engineered tocomprise a nucleic sequence that encodes a rhamnosyl transferase, e.g.,a dTDP-Rha:GlcNAc-UPP α-1,3-rhamnosyltransferase encoded by wbbL. Inanother specific embodiment, provided herein is a prokaryotic host cellcapable of producing O25B, and/or an O25B bioconjugate (i.e., a carrierprotein linked to the E. coli O25B antigen), wherein said host cellnaturally comprises or is engineered to comprise nucleic acids that isat least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ IDNO:11. In another specific embodiment, the prokaryotic host cellcomprises a nucleic acid sequence encoding an oligosaccharyltransferase. In another specific embodiment, the prokaryotic host cellfurther comprises a nucleic acid sequence encoding a carrier proteincomprising a consensus sequence Asn-X-Ser(Thr), wherein X can be anyamino acid except Pro (SEQ ID NO:14); or a carrier protein comprising aconsensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z areindependently selected from any natural amino acid except Pro (SEQ IDNO:15) (see WO 2006/119987). In a specific embodiment, the host cell isE. coli.

In another specific embodiment, provided herein is a prokaryotic hostcell (e.g., a recombinantly engineered prokaryotic host cell) capable ofproducing O25B, and/or an O25B bioconjugate (i.e., a carrier proteinlinked to the E. coli O25B antigen), wherein said host cell naturallycomprises or is engineered to comprise at least one, two, three, four ormore, e.g. all, of the following: (i) a nucleic acid sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:1;(ii) a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical to SEQ ID NO:2; (iii) a nucleic acid sequencethat is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ IDNO:3; (iv) a nucleic acid sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%/, 970/,98%, 99%, or 100% identical to SEQ ID NO:4; (v) a nucleic acid sequencethat is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ IDNO:5; (vi) a nucleic acid sequence that is at least 800/, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89/%, 900/, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identical to SEQ ID NO:6; (vii) a nucleic acidsequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto SEQ ID NO:7; (viii) a nucleic acid sequence that is at least 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:8; (ix) anucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98/%, 99%,or 100% identical to SEQ ID NO:9; (x) a nucleic acid sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10;and/or (xi) a nucleic acid sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90/%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99/%, or 100% identical to SEQ ID NO: 11. In a specific embodiment,said host cell has been engineered to comprise each of said sequences,i.e., said sequences are heterologous to said host cell. In anotherspecific embodiment, the prokaryotic host cell comprises a nucleic acidsequence encoding an oligosaccharyl transferase. In another specificembodiment, the prokaryotic host cell further comprises a nucleic acidsequence encoding a carrier protein comprising a consensus sequenceAsn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ IDNO:14); or a carrier protein comprising a consensus sequenceAsp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selectedfrom any natural amino acid except Pro (SEQ ID NO:15) (see WO2006/119987). In a specific embodiment, the host cell is E. coli.

In another specific embodiment, provided herein is a prokaryotic hostcell (e.g., a recombinantly engineered prokaryotic host cell) thatproduces O25B, wherein said host cell comprises at least two of (i) wbbJ(SEQ ID NO:9) or a nucleic acid that is about or at least 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to or homologous to SEQ IDNO:9; (ii) wbbK (SEQ ID NO: 10) or a nucleic acid that is about or atleast 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to orhomologous to SEQ ID NO:10; and/or (iii) wbbL (SEQ ID NO: 11) or anucleic acid that is about or at least 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identical to or homologous to SEQ ID NO: 11. In anotherspecific embodiment, the prokaryotic host cell comprises a nucleic acidsequence encoding an oligosaccharyl transferase. In another specificembodiment, the prokaryotic host cell further comprises a nucleic acidsequence encoding a carrier protein comprising a consensus sequenceAsn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ IDNO:14); or a carrier protein comprising a consensus sequenceAsp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selectedfrom any natural amino acid except Pro (SEQ ID NO:15) (see WO2006/119987). In a specific embodiment, the host cell is E. coli.

In another specific embodiment, provided herein is a prokaryotic hostcell (e.g., a recombinantly engineered prokaryotic host cell) thatproduces O25B, wherein said host cell comprises each of(i) wbbJ (SEQ IDNO:9) or a nucleic acid that is about or at least 75%, 80%/, 85%, 90%,95%, 96%, 97%, 98%, or 99% identical to or homologous to SEQ ID NO:9;(ii) wbbK (SEQ ID NO: 10) or a nucleic acid that is about or at least75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to orhomologous to SEQ ID NO:10; and (iii) wbbL (SEQ ID NO: 11) or a nucleicacid that is about or at least 75%, 80%, 85%, 90%, 95%, 96%, 97% o, 98%,or 99% identical to or homologous to SEQ ID NO: 11. In another specificembodiment, the prokaryotic host cell comprises a nucleic acid sequenceencoding an oligosaccharyl transferase. In another specific embodiment,the prokaryotic host cell further comprises a nucleic acid sequenceencoding a carrier protein comprising a consensus sequenceAsn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ IDNO:14); or a carrier protein comprising a consensus sequenceAsp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selectedfrom any natural amino acid except Pro (SEQ ID NO:15) (see WO2006/119987). In a specific embodiment, the host cell is E. coli.

In another specific embodiment, provided herein is a prokaryotic hostcell (e.g., a recombinantly engineered prokaryotic host cell) thatproduces O25B, wherein said host cell comprises (i) dTDP-Glucose4,6-dehydratase; (ii) dTDP-6-Deoxy-D-glucose 3,5-epimerase; (iii)Glucose-1-phosphate thymidylyltransferase; (iv) dTDP-4-dehydrorhamnose3,5-epimerase; (v) O antigen flippase; (vi)dTDP-Rha:Glc-Rha(Ac)-GlcNAc-UPP α-1,3-rhamnosyltransferase; (vii)UDP-Glc:Glc-Rha(Ac)-GIcNAc-UPP β-1,6-glucosyltransferase (viii) Oantigen polymerase; (ix) O-acetyl transferase; (x)UDP-Glc:Rha-GlcNAc-UPP α-1,3-glucosyltransferase and/or (xi) dTDP-Rha:GlcNAc-UPP α-1,3-rhamnosyltransferase. In a specific embodiment, theprokaryotic host cell comprises a nucleic acid sequence encoding anoligosaccharyl transferase. In another specific embodiment, theprokaryotic host cell further comprises a nucleic acid sequence encodinga carrier protein comprising a consensus sequence Asn-X-Ser(Thr),wherein X can be any amino acid except Pro (SEQ ID NO:14) or a carrierprotein comprising a consensus sequence or Asp(Glu)-X-Asn-Z-Ser(Thr),wherein X and Z are independently selected from any natural amino acidexcept Pro (SEQ ID NO:15) (see WO 2006/119987). In another specificembodiment, some or all of (i) dTDP-Glucose 4,6-dehydratase; (ii)dTDP-6-Deoxy-D-glucose 3,5-epimerase; (iii) Glucose-1-phosphatethymidylyltransferase; (iv) dTDP-4-dehydrorhamnose 3,5-epimerase; (v) Oantigen flippase; (vi) dTDP-Rha:Glc-Rha(Ac)-GlcNAc-UPPα-1,3-rhamnosyltransferase; (vii) UDP-Glc:Glc-Rha(Ac)-GlcNAc-UPPβ-1,6-glucosyltransferase (viii) O antigen polymerase; (ix) O-acetyltransferase; (x) UDP-Glc:Rha-GlcNAc-UPP α-1,3-glucosyltransferase and/or(xi) dTDP-Rha: GIcNAc-UPP α-1,3-rhamnosyltransferase are heterologous tothe host cell. In another specific embodiment, said oligosaccharyltransferase is heterologous to the host cell. In another specificembodiment, said carrier protein is heterologous to the host cell. In aspecific embodiment, the host cell is E. coli.

In another aspect, provided herein is a prokaryotic host cell (e.g., arecombinantly engineered prokaryotic host cell) that produces E. coliO25A, i.e., said host cell comprises enzymes capable of synthesizing E.coli O025A (see, e.g., FIGS. 3A-3B). In another specific embodiment, theprokaryotic host cell comprises a nucleic acid sequence encoding anoligosaccharyl transferase. In another specific embodiment, theprokaryotic host cell further comprises a nucleic acid sequence encodinga carrier protein comprising a consensus sequence Asn-X-Ser(Thr),wherein X can be any amino acid except Pro (SEQ ID NO:14); or a carrierprotein comprising a consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr),wherein X and Z are independently selected from any natural amino acidexcept Pro (SEQ ID NO:15) (see WO 2006/119987). In a specificembodiment, the host cell is E. coli.

In another aspect, provided herein is a prokaryotic host cell (e.g., arecombinantly engineered prokaryotic host cell) that produces E. coliO1, i.e., said host cell comprises enzymes capable of synthesizing E.coli O1 (see, e.g., FIGS. 12A-12B). In a specific embodiment, providedherein is a host cell that produces E. coli O1A. In a specificembodiment, provided herein is a host cell that produces E. coli O1B. Ina specific embodiment, provided herein is a host cell that produces E.coli O1C. In another specific embodiment, the prokaryotic host cellcomprises a nucleic acid sequence encoding an oligosaccharyltransferase. In another specific embodiment, the prokaryotic host cellfurther comprises a nucleic acid sequence encoding a carrier proteincomprising a consensus sequence Asn-X-Ser(Thr), wherein X can be anyamino acid except Pro (SEQ ID NO:14); or a carrier protein comprising aconsensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z areindependently selected from any natural amino acid except Pro (SEQ IDNO:15) (see WO 2006/119987). In a specific embodiment, the host cell isE. coli.

In another aspect, provided herein is a prokaryotic host cell (e.g., arecombinantly engineered prokaryotic host cell) that produces E. coliO2, i.e., said host cell comprises enzymes capable of synthesizing E.coli O2 (see, e.g., FIGS. 19A-19B). In another specific embodiment, theprokaryotic host cell comprises a nucleic acid sequence encoding anoligosaccharyl transferase. In another specific embodiment, theprokaryotic host cell further comprises a nucleic acid sequence encodinga carrier protein comprising a consensus sequence Asn-X-Ser(Thr),wherein X can be any amino acid except Pro (SEQ ID NO:14); or a carrierprotein comprising a consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr),wherein X and Z are independently selected from any natural amino acidexcept Pro (SEQ ID NO:15) (see WO 2006/119987). In a specificembodiment, the host cell is E. coli.

In another aspect, provided herein is a prokaryotic host cell (e.g., arecombinantly engineered prokaryotic host cell) that produces E. coliO6, i.e., said host cell comprises enzymes capable of synthesizing E.coli O6 (see, e.g., FIGS. 17A-17B). In a specific embodiment, providedherein is a host cell that produces E. coli O6 comprising a branchingGlc monosaccharide or an O6 antigen comprising a branching GlcNAcmonosaccharide. In another specific embodiment, the prokaryotic hostcell comprises a nucleic acid sequence encoding an oligosaccharyltransferase. In another specific embodiment, the prokaryotic host cellfurther comprises a nucleic acid sequence encoding a carrier proteincomprising a consensus sequence Asn-X-Ser(Thr), wherein X can be anyamino acid except Pro (SEQ ID NO: 14); or a carrier protein comprising aconsensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z areindependently selected from any natural amino acid except Pro (SEQ IDNO: 15) (see WO 2006/119987). In a specific embodiment, the host cell isE. coli.

Further provided herein is a prokaryotic host cell (e.g., arecombinantly engineered prokaryotic host cell) capable of producingmore than one type of E. coli O antigen. In a specific embodiment,provided herein is a host cell capable of producing at least two of thefollowing: O25B, O25A, O1 (e.g., O1A, O1B, O1C), O2, and O6. In anotherspecific embodiment, provided herein is a host cell capable of producingO25B and one or more of O25A, O1 (e.g., O1A, O1B, O1C), O2, and O6. Inanother specific embodiment, the prokaryotic host cell comprises anucleic acid sequence encoding an oligosaccharyl transferase. In anotherspecific embodiment, the prokaryotic host cell further comprises anucleic acid sequence encoding a carrier protein comprising a consensussequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQID NO:14); or a carrier protein comprising a consensus sequenceAsp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selectedfrom any natural amino acid except Pro (SEQ ID NO:15) (see WO2006/119987). In a specific embodiment, the host cell is E. coli.

5.3.1 Genetic Background

Any host cells known to those of skill in the art can be used to producethe E. coli O antigens described herein (e.g., O25B) and bioconjugatescomprising the E. coli O antigens described herein (e.g., O25B),including archea, prokaryotic host cells, and eukaryotic host cells.

Exemplary prokaryotic host cells for use in production of the E. coli Oantigens described herein and bioconjugates comprising the E. coli Oantigens described herein include, without limitation, Escherichiaspecies, Shigella species, Klebsiella species, Xhantomonas species,Salmonella species, Yersinia species, Lactococcus species, Lactobacillusspecies, Pseudomonas species, Corynebacterium species, Streptomycesspecies, Streptococcus species, Staphylococcus species, Bacillusspecies, and Clostridium species. In a specific embodiment, the hostcell used to produce the E. coli O antigens described herein andbioconjugates comprising the E. coli 0 antigens described herein is E.coli.

In certain embodiments, the host cells used to produce the E. coli Oantigens and bioconjugates described herein are engineered to compriseheterologous nucleic acids, e.g., heterologous nucleic acids that encodeone or more carrier proteins and/or heterologous nucleic acids thatencode one or more proteins, e.g., genes encoding one or more proteins.In a specific embodiment, heterologous nucleic acids that encodeproteins involved in glycosylation pathways (e.g., prokaryotic and/oreukaryotic glycosylation pathways) may be introduced into the host cellsdescribed herein. Such nucleic acids may encode proteins including,without limitation, oligosaccharyl transferases and/orglycosyltransferases. Heterologous nucleic acids (e.g., nucleic acidsthat encode carrier proteins and/or nucleic acids that encode otherproteins, e.g., proteins involved in glycosylation) can be introducedinto the host cells described herein using any methods known to those ofskill in the art, e.g., electroporation, chemical transformation by heatshock, natural transformation, phage transduction, and conjugation. Inspecific embodiments, heterologous nucleic acids are introduced into thehost cells described herein using a plasmid, e.g., the heterologousnucleic acids are expressed in the host cells by a plasmid (e.g., anexpression vector). In another specific embodiment, heterologous nucleicacids are introduced into the host cells described herein using themethod of insertion described in International Patent application No.PCT/EP2013/068737.

In certain embodiments, additional modifications may be introduced(e.g., using recombinant techniques) into the host cells describedherein. For example, host cell nucleic acids (e.g., genes) that encodeproteins that form part of a possibly competing or interferingglycosylation pathway (e.g., compete or interfere with one or moreheterologous genes involved in glycosylation that are recombinantlyintroduced into the host cell) can be deleted or modified in the hostcell background (genome) in a manner that makes theminactive/dysfunctional (i.e., the host cell nucleic acids that aredeleted/modified do not encode a functional protein or do not encode aprotein whatsoever). In certain embodiments, when nucleic acids aredeleted from the genome of the host cells provided herein, they arereplaced by a desirable sequence, e.g., a sequence that is useful forglycoprotein production.

Exemplary genes that can be deleted in host cells (and, in some cases,replaced with other desired nucleic acid sequences) include genes ofhost cells involved in glycolipid biosynthesis, such as waaL (see, e.g.,Feldman et al., 2005, PNAS USA 102:3016-3021), the lipid A corebiosynthesis cluster (waa), galactose cluster (gal), arabinose cluster(ara), colonic acid cluster (wc), capsular polysaccharide cluster,undecaprenol-p biosynthesis genes (e.g. uppS, uppP), und-P recyclinggenes, metabolic enzymes involved in nucleotide activated sugarbiosynthesis, enterobacterial common antigen cluster, and prophage Oantigen modification clusters like the gtrABS cluster. In a specificembodiment, the host cells described herein are modified such that theydo not produce any O antigens other than a desired O antigen from anExPEC, e.g., O25B. In a specific embodiment, one or more of the waaLgene, girA gene, gtrB gene, gtrS gene, or the rb gene cluster is deletedor functionally inactivated from the genome of a prokaryotic host cellprovided herein. In one embodiment, a host cell used herein is E. colithat produces O25B antigen, wherein the waaL gene, gtrA gene, gtrB gene,and gtrS gene are deleted or functionally inactivated from the genome ofthe host cell. In another embodiment, a host cell used herein is E. colithat produces O25B antigen, wherein the waaL gene and gtrS gene aredeleted or functionally inactivated from the genome of the host cell.

In certain embodiments, the modified host cells provided herein can beused for protein glycosylation. Protein glycosylation may be designed toproduce bioconjugates for use in vaccine formulations, e.g., vaccinesthat contain E. coli polysaccharide antigen(s), e.g., O25 (e.g., O25B),O1, O2, and O6.

5.3.2 Carrier Proteins

Any carrier protein suitable for use in the production of conjugatevaccines (e.g., bioconjugates for use in vaccines) can be used herein,e.g., nucleic acids encoding the carrier protein can be introduced intoa host provided herein for the production of a bioconjugate comprising acarrier protein linked to an ExPEC antigen (e.g., O25B). Exemplarycarrier proteins include, without limitation, detoxified Exotoxin A ofP. aeruginosa (EPA; see, e.g., Ihssen, et al., (2010) Microbial cellfactories 9, 61), CRM 197, maltose binding protein (MBP), Diphtheriatoxoid, Tetanus toxoid, detoxified hemolysin A of S. aureus, clumpingfactor A, clumping factor B, E. coli FimH, E. coli FimHC, E. coli heatlabile enterotoxin, detoxified variants of E. coli heat labileenterotoxin, Cholera toxin B subunit (CTB), cholera toxin, detoxifiedvariants of cholera toxin, E. coli Sat protein, the passenger domain ofE. coli Sat protein, Streptococcus pneumoniae Pneumolysin and detoxifiedvariants thereof, C. jejuni AcrA, and C. jejuni natural glycoproteins.For EPA, various detoxified protein variants have been described inliterature and could be used as carrier proteins.

In certain embodiments, the carrier proteins used in the generation ofthe bioconjugates described herein are modified, e.g., modified in sucha way that the protein is less toxic and/or more susceptible toglycosylation. In a specific embodiment, the carrier proteins used inthe generation of the bioconjugates described herein are modified suchthat the number of glycosylation sites in the carrier proteins ismaximized in a manner that allows for lower concentrations of theprotein to be administered, e.g., in an immunogenic composition, in itsbioconjugate form.

In certain embodiments, the carrier proteins described herein aremodified to include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more glycosylationsites than would normally be associated with the carrier protein (e.g.,relative to the number of glycosylation sites associated with thecarrier protein in its native/natural, e.g., “wild-type” state). Inspecific embodiments, introduction of glycosylation sites isaccomplished by insertion of glycosylation consensus sequences (e.g.,Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO:14); or Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independentlyselected from any natural amino acid except Pro (SEQ ID NO:15) (see WO2006/119987)) anywhere in the primary structure of the protein.Introduction of such glycosylation sites can be accomplished by, e.g.,adding new amino acids to the primary structure of the protein (i.e.,the glycosylation sites are added, in full or in part), or by mutatingexisting amino acids in the protein in order to generate theglycosylation sites (i.e., amino acids are not added to the protein, butselected amino acids of the protein are mutated so as to formglycosylation sites). Those of skill in the art will recognize that theamino acid sequence of a protein can be readily modified usingapproaches known in the art, e.g., recombinant approaches that includemodification of the nucleic acid sequence encoding the protein. Inspecific embodiments, glycosylation consensus sequences are introducedinto specific regions of the carrier protein, e.g., surface structuresof the protein, at the N or C termini of the protein, and/or in loopsthat are stabilized by disulfide bridges at the base of the protein.

In certain embodiments, the classical 5 amino acid glycosylationconsensus sequence may be extended by lysine residues for more efficientglycosylation, and thus the inserted consensus sequence may encode 5, 6,or 7 amino acids that should be inserted or that replace acceptorprotein amino acids. In one particular embodiment a carrier protein isdetoxified EPA comprising 4 consensus glycosylation sequencesAsp/Glu-X-Asn-Z-Ser/Thr (SEQ ID NO: 15), and has an amino acid sequenceas provided in SEQ ID NO: 13.

In certain embodiments, the carrier proteins used in the generation ofthe bioconjugates described herein comprise a “tag,” i.e., a sequence ofamino acids that allows for the isolation and/or identification of thecarrier protein. For example, adding a tag to a carrier proteindescribed herein can be useful in the purification of that protein and,hence, the purification of conjugate vaccines comprising the taggedcarrier protein. Exemplary tags that can be used herein include, withoutlimitation, histidine (HIS) tags (e.g., hexa histidine-tag, or6XHis-Tag), FLAG-TAG, and HA tags. In certain embodiments, the tags usedherein are removable, e.g., removal by chemical agents or by enzymaticmeans, once they are no longer needed, e.g., after the protein has beenpurified.

In certain embodiments, the carrier proteins described herein comprise asignal sequence that targets the carrier protein to the periplasmicspace of the host cell that expresses the carrier protein. In a specificembodiment, the signal sequence is from E. coli DsbA, E. coli outermembrane porin A (OmpA), E. coli maltose binding protein (MalE), Erwiniacarotovorans pectate lyase (PelB), FlgI, NikA, or Bacillus sp.endoxylanase (XynA), heat labile E. coli enterotoxin LTIIb, Bacillusendoxylanase XynA, or E. coli flagellin (FlgI).

5.3.3 Glycosylation Machinery

The host cells provided herein comprise, and/or can be modified tocomprise, nucleic acids that encode genetic machinery (e.g.,glycosyltransferases) capable of producing O antigens from ExPEC, e.g.,the O25 (e.g., O25B), O1, O2, and/or O6 antigens. See Section 5.1.

Glycosyltransferases

The host cells provided herein comprise nucleic acids that encodeglycosyltransferases capable of producing ExPEC O antigens, e.g., an Oantigen from E. coli of serotype O25 (e.g., O25A or O25B, see FIG. 3B),O1 (see FIGS. 12A and 12B), O2 (see FIGS. 19A-19B), and O6 (e.g., an O6serotype producing an O6 antigen comprising a branching Glcmonosaccharide or an O6 antigen comprising a branching GlcNAcmonosaccharide, see FIGS. 17A-17B). Exemplary nucleic acids aredescribed in Section 5.1. In certain embodiments, some or all of thenucleic acids that encode glycosyltransferases capable of producing anExPEC O antigen are naturally expressed by the host cells providedherein (e.g., the nucleic acids are present in the “wild-type”background of the host cell). In certain embodiments, some or all of thenucleic acids that encode glycosyltransferases capable of producing anExPEC O antigen are not naturally expressed by the host cells providedherein, i.e., some or all of the nucleic acids are heterologous to thehost cell. Host cells can be engineered to comprise specific nucleicacids, e.g., the nucleic acids described in Section 5.1, using methodsknown in the art, e.g., the methods described in Section 5.3.

In a specific embodiment, the host cells provided herein comprisenucleic acids that encode glycosyltransferases capable of producing anE. coli O antigen of the O25B serotype, i.e., an O25B antigen describedherein. In a specific embodiment, said nucleic acids encode the ribcluster from upec138 (SEQ ID NO: 12), or a gene cluster that is about orat least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to orhomologous to SEQ ID NO: 12.

In another specific embodiment, said nucleic acids encode the rfbcluster from upec163, or a gene cluster that is about or at least 75%,80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to or homologous tothe rfb cluster from upec163. In another specific embodiment, saidnucleic acids encode the rfb cluster from upec177, or a gene clusterthat is about or at least 75%, 80%, 85%, 90/%, 95%, 96%, 97%, 98% or 99%identical to or homologous to the rjb cluster from upec177.

In another specific embodiment, said nucleic acids that encodeglycosyltransferases capable of producing an E. coli O antigen of theO25B serotype are genes of an O25B serotype, wherein said genes are rmlB(SEQ ID NO: 1), rmlD (SEQ ID NO:2) rmL4 (SEQ ID NO:3), rmlC (SEQ IDNO:4), wzx (SEQ ID NO:5), wekA (SEQ ID NO:6), wekB (SEQ ID NO:7), wzy(SEQ ID NO:8), wbbJ (SEQ ID NO:9), wbbK (SEQ ID NO:10), and wbbL (SEQ IDNO:11). See tables 3 and 9.

In a specific embodiment, the host cells provided herein comprisenucleic acids that encode proteins (e.g., glycosyltransferases) capableof producing an E. coli O antigen of the O25A serotype, i.e., an O25Aantigen described herein. In another specific embodiment, said nucleicacids that encode proteins (e.g., glycosyltransferases) capable ofproducing an E. coli O antigen of the O25A serotype are genes of an O25serotype, wherein said genes are rmlB, rmlD, rmlA, rmlC, wzx, wekA,wekB, wekC, wzy, fnlA, fnlB, fnlC, wbuB, and/or wbuC. See Wang, et al.(2010) J Clin Microbiol 48, 2066-2074; GenBank GU014554; and Table 2.

In another specific embodiment, the host cells provided herein comprisenucleic acids that encode glycosyltransferases capable of producing an Oantigen E. coli of the O2 serotype.

In another specific embodiment, said nucleic acids that encodeglycosyltransferases capable of producing an E. coli O antigen of the O2serotype are genes of an O2 serotype, wherein said genes are rmlB, rmlD,rmlA, rmlC, wza, wekP, wekQ, wekR, wzy, fdtA, fdtB, and/or fdtC. See Li,et al., (2010) J Microbiol Methods 82, 71-77; Fratamico, et al., 2010,Canadian Journal of Microbiology 56, 308-316; and Table 5.

In another specific embodiment, the host cells provided herein comprisenucleic acids that encode glycosyltransferases capable of producing an Oantigen E. coli of the O6 serotype. See Welch et al., 2002, PNAS USA99(26):17020-17024; Jann et al., Carbohydr. Res. 263 (1994) 217-225, andJansson et al., Carbohydr. Res. 131 (1984) 277-283. In a specificembodiment, said O6 serotype comprises a branched Glc monosaccharide.

In another specific embodiment, the host cells provided herein comprisenucleic acids that encode glycosyltransferases capable of producing an Oantigen E. coli of the O1 serotype. In a specific embodiment, said O1serotype is O1A. In another specific embodiment, said nucleic acids thatencode glycosyltransferases capable of producing an E. coli O antigen ofthe O1 serotype are genes of an O1 serotype, wherein said genes arermlB, rmlD, rmlA, rmlC, wzx, mnaA, wekM, wzy, wekN, and/or wekO.

Oligosaccharyl Transferases

Oligosaccharyl transferases transfer lipid-linked oligosaccharides toasparagine residues of nascent polypeptide chains that comprise anN-glycoxylation consensus motif, e.g., Asn-X-Ser(Thr), wherein X can beany amino acid except Pro (SEQ ID NO:14); or Asp(Glu)-X-Asn-Z-Ser(Thr),wherein X and Z are independently selected from any natural amino acidexcept Pro (SEQ ID NO:15) (see WO 2006/119987). See, e.g., WO2003/074687 and WO 2006/119987, the disclosures of which are hereinincorporated by reference in their entirety.

In certain embodiments, the host cells provided herein comprise anucleic acid that encodes an oligosaccharyl transferase. The nucleicacid that encodes an oligosaccharyl transferase can be native to thehost cell, or can be introduced into the host cell using geneticapproaches, as described above. The oligosaccharyl transferase can befrom any source known in the art. In a specific embodiment, theoligosaccharyl transferase is an oligosaccharyl transferase fromCampylobacter. In another specific embodiment, the oligosaccharyltransferase is an oligosaccharyl transferase from Campylobacter jejuni(i.e., pglB; see, e.g., Wacker et al., 2002, Science 298:1790-1793; seealso, e.g., NCBI Gene ID: 3231775, UniProt Accession No. O86154). Inanother specific embodiment, the oligosaccharyl transferase is anoligosaccharyl transferase from Campylobacter lari (see, e.g., NCBI GeneID: 7410986).

5.4 Bloconjugates

In certain embodiments, the host cells described herein can be used toproduce bioconjugates comprising an E. coli O antigen described herein(e.g., O25B; see Section 5.2) linked to a carrier protein. Methods ofproducing such bioconjugates using host cells are known in the art. See,e.g., WO 2003/074687 and WO 2006/119987.

Alternatively, glycoconjugates can be prepared by chemical synthesis,i.e., prepared outside of host cells (in vitro). For example, the E.coli O antigens described herein, e.g., O25B, can be conjugated tocarrier proteins using methods known to those of skill in the art,including by means of using activation reactive groups in thepolysaccharide/oligosaccharide as well as the protein carrier. See,e.g., Pawlowski et al., 2000, Vaccine 18:1873-1885; and Robbins, et al.,2009, Proc Natl Acad Sci USA 106:7974-7978), the disclosures of whichare herein incorporated by reference. Such approaches compriseextraction of antigenic polysaccharides/oligosaccharides from hostcells, purifying the polysaccharides/oligosaccharides, chemicallyactivating the polysaccharides/oligosaccharides, and conjugating thepolysaccharides/oligosaccharides to a carrier protein.

Bioconjugates, as described herein, have advantageous properties overglycoconjugates, e.g., bioconjugates require less chemicals inmanufacture and are more consistent in terms of the final productgenerated. Thus, bioconjugates are preferred over chemically producedglycoconjugates.

In a specific embodiment, provided herein are bioconjugates comprising acarrier protein linked to an ExPEC O antigen described herein. SeeSection 5.2.

In a specific embodiment, provided herein is a bioconjugate comprising acarrier protein (e.g., EPA) N-linked to E. coli O25B.

In another specific embodiment, provided herein is a bioconjugatecomprising a carrier protein (e.g., EPA) linked to a compound of FormulaO25B presented below:

wherein n is an integer between 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25,20 to 25, 10 to 20, or 15 to 20. In a specific embodiment, the carrierprotein is N-linked to the O antigen of Formula O25B, i.e., O25B islinked to the Asn residue of a carrier protein comprising the sequenceAsn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ IDNO:14); or a carrier protein comprising a consensus sequenceAsp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selectedfrom any natural amino acid except Pro (SEQ ID NO: 15).

In another specific embodiment, provided herein is a bioconjugatecomprising a carrier protein (e.g., EPA) linked to a compound of FormulaO25B′, presented below:

wherein n is an integer between 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25,20 to 25, 10 to 20, or 15 to 20. In a specific embodiment, the carrierprotein is N-linked to the O antigen of Formula O25B′.

In another specific embodiment, provided herein is a bioconjugatecomprising a carrier protein (e.g., EPA) linked to an O25A antigen. In aspecific embodiment, said O25A antigen comprises the formula O25A:

In another specific embodiment, provided herein is a bioconjugatecomprising a carrier protein (e.g., EPA) linked to an O1 antigen. In aspecific embodiment, said O1 antigen is O1A, e.g., said antigencomprises the formula OIA:

In another specific embodiment, said O1 antigen is O1B, e.g., saidantigen comprises the formula O1B:

In another specific embodiment, said O1 antigen is O1C, e.g., saidantigen comprises the formula O1C:

In another specific embodiment, provided herein is a bioconjugatecomprising a carrier protein (e.g., EPA) linked to an O2 antigen. In aspecific embodiment, said O2 antigen comprises the formula O2:

In another specific embodiment, provided herein is a bioconjugatecomprising a carrier protein (e.g., EPA) linked to an O6 antigen. In aspecific embodiment, said O6 antigen comprises the formula O6Glc:

The bioconjugates described herein can be purified by any method knownin the art for purification of a protein, for example, by chromatography(e.g., ion exchange, anionic exchange, affinity, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. See, e.g.,Saraswat et al., 2013, Biomed. Res. Int. ID #312709 (p. 1-18); see alsothe methods described in WO 2009/104074. Further, the bioconjugates maybe fused to heterologous polypeptide sequences described herein orotherwise known in the art to facilitate purification. The actualconditions used to purify a particular bioconjugate will depend, inpart, on the synthesis strategy (e.g., synthetic production vs.recombinant production) and on factors such as net charge,hydrophobicity, and/or hydrophilicity of the bioconjugate, and will beapparent to those having skill in the art.

5.5 Antibodies Against O25B

The O25B antigen described herein (see Section 5.2) and/or bioconjugatescomprising the O25B antigen described herein (see Section 5.4) can beused to elicit neutralizing antibodies against ExPEC. In a specificembodiment, the O25B antigen described herein and/or bioconjugatescomprising the O25B antigen described herein can be administered to asubject (e.g., a human, mouse, rabbit, rat, guinea pig, etc.) to inducean immune response that includes the production of antibodies. Suchantibodies can be isolated using techniques known to one of skill in theart (e.g., immunoaffinity chromatography, centrifugation, precipitation,etc.).

In addition, the O25B antigen described herein can be used to screen forantibodies from antibody libraries. For example, isolated O25B can beimmobilized to a solid support (e.g., a silica gel, a resin, aderivatized plastic film, a glass bead, cotton, a plastic bead, apolystyrene bead, an alumina gel, or a polysaccharide, a magnetic bead),and screened for binding to antibodies. As an alternative, antibodies tobe screened may be immobilized to a solid support and screened forbinding to O25B. Any screening assay, such as a panning assay, ELISA,surface plasmon resonance, or other antibody screening assay known inthe art may be used to screen for antibodies that bind to O25B. Theantibody library screened may be a commercially available antibodylibrary, an in vitro generated library, or a library obtained byidentifying and cloning or isolating antibodies from an individualinfected with EXPEC. Antibody libraries may be generated in accordancewith methods known in the art. In a particular embodiment, the antibodylibrary is generated by cloning the antibodies and using them in phagedisplay libraries or a phagemid display library.

Antibodies identified or elicited using O25B and/or a bioconjugate ofO25B can include immunoglobulin molecules and immunologically activeportions of immunoglobulin molecules, i.e., molecules that contain anantigen binding site that specifically binds to O25B. The immunoglobulinmolecules may be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY),class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ and IgA₂) or subclass ofimmunoglobulin molecule. Antibodies include, but are not limited to,monoclonal antibodies, multispecific antibodies, human antibodies,humanized antibodies, chimeric antibodies, single-chain Fvs (scFv),single chain antibodies, Fab fragments, F(ab) fragments,disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies(including, e.g., anti-Id antibodies to antibodies elicited oridentified using a method described herein), and epitope-bindingfragments of any of the above. In a specific embodiment, an antibodyelicited or identified using O25B and/or a bioconjugate of O25B is ahuman or humanized monoclonal antibody.

Antibodies elicited or identified using using O25B and/or a bioconjugateof O25B can be used to monitor the efficacy of a therapy and/or diseaseprogression. Any immunoassay system known in the art may be used forthis purpose including, but not limited to, competitive andnoncompetitive assay systems using techniques such as radioimmunoassays,ELISA (enzyme linked immunosorbent assays), “sandwich” immunoassays,precipitin reactions, gel diffusion precipitin reactions,immunodiffusion assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays and immunoelectrophoresis assays.

Antibodies elicited or identified using O25B and/or a bioconjugate ofO25B can be used to detect E. coli O25B strains, for example, from aplurality of E. coli strains and/or to diagnose an infection by an E.coli O25B strain.

5.6 Compositions

5.6.1 Compositions Comprising Host Cells

In one aspect, provided herein are compositions comprising the hostcells described herein. Such compositions can be used in methods forgenerating the bioconjugates described herein (see Section 5.4), e.g.,the compositions can be cultured under conditions suitable for theproduction of proteins. Subsequently, bioconjugates can be isolated fromsaid compositions using methods known in the art.

The compositions comprising the host cells provided herein can compriseadditional components suitable for maintenance and survival of the hostcells described herein, and can additionally comprise additionalcomponents required or beneficial to the production of proteins by thehost cells, e.g., inducers for inducible promoters, such as arabinose,IPTG.

5.6.2 Compositions Comprising Antigens and/or Bioconjugates

In another aspect, provided herein are compositions (e.g.,pharmaceutical compositions) comprising one or more of the E. coli Oantigens described herein (see Section 5.2) and compositions (e.g.,pharmaceutical compositions) comprising one or more of the bioconjugatesdescribed herein (see Section 5.4). In a specific embodiment, acomposition provided herein comprises one or more of the E. coli Oantigens described herein (see Section 5.2). In another specificembodiment, a composition provided herein comprises one or more of thebioconjugates described herein (see Section 5.4). In another specificembodiment, a composition provided herein comprises one or more of theE. coli O antigens described herein (see Section 5.2) and one or more ofthe bioconjugates described herein (see Section 5.4). The compositionsdescribed herein are useful in the treatment and prevention of infectionof subjects (e.g., human subjects) with extra-intestinal pathogenic E.coli (ExPEC). See Section 5.7.

In certain embodiments, in addition to comprising an E. coli O antigendescribed herein (see Section 5.2) and/or a bioconjugate describedherein (see Section 5.4), the compositions (e.g., pharmaceuticalcompositions) described herein comprise a pharmaceutically acceptablecarrier. As used herein, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeiae for use in animals, and more particularly in humans. Theterm “carrier,” as used herein in the context of a pharmaceuticallyacceptable carrier, refers to a diluent, adjuvant, excipient, or vehiclewith which the pharmaceutical composition is administered. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Suitable excipients include starch, glucose, lactose, sucrose, gelatin,malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmonostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. Examples of suitablepharmaceutical carriers are described in “Remington's PharmaceuticalSciences” by E. W. Martin.

In a specific embodiment, provided herein is a composition comprising acarrier protein (e.g., a carrier protein described in Section 5.3.2)linked to an antigen described herein, e.g., an ExPEC O antigendescribed in Section 5.2.

In another specific embodiment, a composition provided herein comprisesa carrier protein (e.g., a carrier protein described in Section 5.3.2,e.g., EPA or MBP) linked to E. coli O25B (see Section 5.2).

In another specific embodiment, a composition provided herein comprisesa carrier protein (e.g., a carrier protein described in Section 5.3.2,e.g., EPA or MBP) linked to E. coli 025A (see Section 5.2).

In another specific embodiment, a composition provided herein comprisesa carrier protein (e.g., a carrier protein described in Section 5.3.2,e.g., EPA or MBP) linked to E. coli O1 (see Section 5.2). In anotherspecific embodiment, said O1 macromolecule is O1A. In another specificembodiment, said O1 macromolecule is O1B. In another specificembodiment, said O1 macromolecule is O1C.

In another specific embodiment, a composition provided herein comprisesa carrier protein (e.g., a carrier protein described in Section 5.3.2,e.g., EPA or MBP) linked to E. coli O2 (see Section 5.2).

In another specific embodiment, a composition provided herein comprisesa carrier protein (e.g., a carrier protein described in Section 5.3.2,e.g., EPA or MBP) linked to E. coli O6 (see Section 5.2). In a specificembodiment, said O6 macromolecule is an O6 macromolecule comprising abranching Glc monosaccharide.

In another specific embodiment, provided herein is a composition, e.g.,a pharmaceutical composition, comprising (i) an O25 (e.g., O25A or O25B)macromolecule, or a bioconjugate comprising O25 (e.g., O25A or O25B) and(ii) an O1 macromolecule or a bioconjugate comprising O1. See Section5.2. In a specific embodiment, said O25 macromolecule is an O25Bmacromolecule. In another specific embodiment, said O1 macromolecule isO1A. In another specific embodiment, said O1 macromolecule is O1B. Inanother specific embodiment, said O1 macromolecule is O1C.

In another specific embodiment, provided herein is a composition, e.g.,a pharmaceutical composition, comprising (i) an O25 (e.g., O25A or O25B)macromolecule, or a bioconjugate comprising O25 (e.g., O25A or O25B) and(ii) an O2 macromolecule or a bioconjugate comprising O2. See Sections5.2 and 5.4. In a specific embodiment, said O25 macromolecule is an O25Bmacromolecule.

In another specific embodiment, provided herein is a composition, e.g.,a pharmaceutical composition, comprising (i) an O25 (e.g., O25A or O25B)macromolecule, or a bioconjugate comprising O25 (e.g., O25A or O25B) and(ii) an O6 macromolecule (e.g., a O6 macromolecule comprising abranching Glc monosaccharide or a branching GlcNAc monosaccharide) or abioconjugate comprising O6. See Sections 5.2 and 5.4. In a specificembodiment, said O25 macromolecule is an O25B macromolecule. In anotherspecific embodiment, said O6 macromolecule is an O6 macromoleculecomprising a branching Glc monosaccharide.

In another specific embodiment, provided herein is a composition, e.g.,a pharmaceutical composition, comprising E. coli O25B (see Section 5.2)or a or a bioconjugate comprising O25 (see Section 5.4) and at least oneof the following: (i) E. coli O1 or a bioconjugate comprising O1 (seeSections 5.2 and 5.4); (ii) E. coli O2 or a bioconjugate comprising O2(see Sections 5.2 and 5.4); and/or (iii) E. coli O6 or a bioconjugatecomprising O6 (see Sections 5.2 and 5.4). In another specificembodiment, said O1 is O1A. In another specific embodiment, said O1 isO1B. In another specific embodiment, said O1 is O1C. In another specificembodiment, said O6 is O6 comprising a branching Gic monosaccharide.

In another specific embodiment, provided herein is a composition, e.g.,a pharmaceutical composition, comprising at least two of the following:(i) an O25 (e.g., O25A or O25B) macromolecule or a bioconjugatecomprising O25 (e.g., O25A or O25B); (ii) an O1 macromolecule or abioconjugate comprising O1; (iii) an O2 macromolecule or a bioconjugatecomprising O2; and/or (iv) an O6 macromolecule (e.g., a O6 macromoleculecomprising a branching Glc monosaccharide or a branching GlcNAcmonosaccharide) or a bioconjugate comprising O6. In a specificembodiment, said O25 macromolecule is an O25B macromolecule.

In another specific embodiment, said O1 macromolecule is O1A. In anotherspecific embodiment, said O1 macromolecule is O1B. In another specificembodiment, said O1 macromolecule is O1C. In another specificembodiment, said O6 macromolecule is an O6 macromolecule comprising abranching Glc monosaccharide.

In another specific embodiment, provided herein is a composition, e.g.,a pharmaceutical composition, comprising a bioconjugate comprising E.coli O25B and a bioconjugate comprising E. coli O1A. Such bioconjugatesare described in Section 5.4.

In another specific embodiment, provided herein is a composition, e.g.,a pharmaceutical composition, comprising a bioconjugate comprising E.coli O25B and a bioconjugate comprising E. coli O1B. Such bioconjugatesare described in Section 5.4.

In another specific embodiment, provided herein is a composition, e.g.,a pharmaceutical composition, comprising a bioconjugate comprising E.coli O25B and a bioconjugate comprising E. coli O1C. Such bioconjugatesare described in Section 5.4.

In another specific embodiment, provided herein is a composition, e.g.,a pharmaceutical composition, comprising a bioconjugate comprising E.coli O25B and a bioconjugate comprising E. coli O2. Such bioconjugatesare described in Section 5.4.

In another specific embodiment, provided herein is a composition, e.g.,a pharmaceutical composition, comprising a bioconjugate comprising E.coli O25B and a bioconjugate comprising E. coli O6. Such bioconjugatesare described in Section 5.4.

In another specific embodiment, a composition provided herein comprisesa carrier protein (e.g., a carrier protein described in Section 5.3.2,e.g., EPA or MBP) linked to E. coli O25B (see Section 5.2), (ii) acarrier protein (e.g., a carrier protein described in Section 5.3.2,e.g., EPA or MBP) linked to an E. coli O antigen of the O1 serotype,e.g., O1A (see Section 5.2), (iii) a carrier protein (e.g., a carrierprotein described in Section 5.1.2, e.g., EPA or MBP) linked to an E.coli O antigen of the O2 serotype (see Section 5.2), and (iv) a carrierprotein (e.g., a carrier protein described in Section 5.3.2, e.g., EPAor MBP) linked to an E. coli O antigen of the O6 serotype (see Section5.2).

In certain embodiments, the foregoing compositions comprise a carrierprotein (e.g., a carrier protein described in Section 5.3.2, e.g., EPAor MBP) linked to an E. coli O antigen of an E. coli serotype other thanO1, O2, O6, or O25. Other useful E. coli serotypes are described, e.g.,in Example 1 and Table 1, below.

In another specific embodiment, a composition provided herein comprisesan O25 (e.g., O25A or O25B) macromolecule.

In another specific embodiment, a composition provided herein comprisesan 01 macromolecule (e.g., O1A, O1B, or O1C).

In another specific embodiment, a composition provided herein comprisesan O2 macromolecule.

In another specific embodiment, a composition provided herein comprisesan O6 macromolecule (e.g., a O6 macromolecule comprising a branching Glcmonosaccharide or a branching GlcNAc monosaccharide).

In another specific embodiment, a composition provided herein comprisesan O25 (e.g., O25A or O25B) macromolecule, an O1 macromolecule, an O2macromolecule, and an O6 macromolecule (e.g., a O6 macromoleculecomprising a branching Glc monosaccharide or a branching GlcNAcmonosaccharide). In a specific embodiment, said O25 macromolecule is anO25B macromolecule. In another specific embodiment, said O1macromolecule is O1A. In another specific embodiment, said O6macromolecule is an O6 macromolecule comprising a branching Glcmonosaccharide.

The compositions provided herein can be used for eliciting an immuneresponse in a host to whom the composition is administered, i.e., areimmunogenic. Thus, the compositions described herein can be used asvaccines against ExPEC infection, or can be used in the treatment ofExPEC infection, and can accordingly be formulated as pharmaceuticalcompositions. See Section 5.7.

The compositions comprising the bioconjugates and/or macromoleculesdescribed herein may comprise any additional components suitable for usein pharmaceutical administration. In specific embodiments, thecompositions described herein are monovalent formulations. In otherembodiments, the compositions described herein are multivalentformulations, e.g., bivalent, trivalent, and tetravalent formulations.For example, a multivalent formulation comprises more than onebioconjugate or E. coli O antigen described herein. See Sections 5.2 and5.4 for description of E. coli O antigens and bioconjugates,respectively. In a specific embodiment, a composition described hereinis a tetravalent formulation comprising a macromolecule or bioconjugate,wherein said valences are from E. coli O antigens of the O25B, O1A, O6,and O2 serotypes/subserotypes.

In certain embodiments, the compositions described herein additionallycomprise a preservative, e.g., the mercury derivative thimerosal. In aspecific embodiment, the pharmaceutical compositions described hereincomprise 0.001% to 0.01% thimerosal. In other embodiments, thepharmaceutical compositions described herein do not comprise apreservative.

In certain embodiments, the compositions described herein (e.g., theimmunogenic compositions) comprise, or are administered in combinationwith, an adjuvant. The adjuvant for administration in combination with acomposition described herein may be administered before, concomitantlywith, or after administration of said composition. In some embodiments,the term “adjuvant” refers to a compound that when administered inconjunction with or as part of a composition described herein augments,enhances and/or boosts the immune response to a bioconjugate, but whenthe compound is administered alone does not generate an immune responseto the bioconjugate. In some embodiments, the adjuvant generates animmune response to the poly bioconjugate peptide and does not produce anallergy or other adverse reaction. Adjuvants can enhance an immuneresponse by several mechanisms including, e.g., lymphocyte recruitment,stimulation of B and/or T cells, and stimulation of macrophages.

Specific examples of adjuvants include, but are not limited to, aluminumsalts (alum) (such as aluminum hydroxide, aluminum phosphate, andaluminum sulfate), 3 De-O-acylated monophosphoryl lipid A (MPL) (seeUnited Kingdom Patent GB2220211), MF59 (Novartis), AS03(GlaxoSmithKline), AS04 (GlaxoSmithKline), polysorbate 80 (Tween 80; ICLAmericas, Inc.), imidazopyridine compounds (see InternationalApplication No. PCT/US2007/064857, published as InternationalPublication No. WO2007/109812), imidazoquinoxaline compounds (seeInternational Application No. PCT/US2007/064858, published asInternational Publication No. WO2007/109813) and saponins, such as QS21(see Kensil et al., in Vaccine Design: The Subunit and Adjuvant Approach(eds. Powell & Newman, Plenum Press, N Y, 1995); U.S. Pat. No.5,057,540). In some embodiments, the adjuvant is Freund's adjuvant(complete or incomplete). Other adjuvants are oil in water emulsions(such as squalene or peanut oil), optionally in combination with immunestimulants, such as monophosphoryl lipid A (see Stoute et al., N. Engl.J. Med. 336, 86-91 (1997)). Another adjuvant is CpG (Bioworld Today,Nov. 15, 1998).

In certain embodiments, the compositions described herein are formulatedto be suitable for the intended route of administration to a subject.For example, the compositions described herein may be formulated to besuitable for subcutaneous, parenteral, oral, intradermal, transdermal,colorectal, intraperitoneal, and rectal administration. In a specificembodiment, the pharmaceutical composition may be formulated forintravenous, oral, intraperitoneal, intranasal, intratracheal,subcutaneous, intramuscular, topical, intradermal, transdermal orpulmonary administration.

In certain embodiments, the compositions described herein additionallycomprise one or more buffers, e.g., phosphate buffer and sucrosephosphate glutamate buffer. In other embodiments, the compositionsdescribed herein do not comprise buffers.

In certain embodiments, the compositions described herein additionallycomprise one or more salts, e.g., sodium chloride, calcium chloride,sodium phosphate, monosodium glutamate, and aluminum salts (e.g.,aluminum hydroxide, aluminum phosphate, alum (potassium aluminumsulfate), or a mixture of such aluminum salts). In other embodiments,the compositions described herein do not comprise salts.

The compositions described herein can be included in a container, pack,or dispenser together with instructions for administration.

The compositions described herein can be stored before use, e.g., thecompositions can be stored frozen (e.g., at about −20° C. or at about−70° C.); stored in refrigerated conditions (e.g., at about 4° C.); orstored at room temperature.

5.7 Prophylactic and Therapeutic Uses

Provided herein are methods of treating and preventing extraintestinalE. coli (ExPEC) infection of a subject comprising administering to thesubject an E. coli O antigen described herein (see Section 5.2), abioconjugate described herein (see Section 5.4), or a compositiondescribed herein (see Section 5.6.2). In a specific embodiment, thecompositions described herein (see Section 5.6.2) are used in theprevention of infection of a subject (e.g., human subjects) by ExPEC,i.e., the compositions described herein are used to vaccinate a subjectagainst ExPEC infection. In another specific embodiment, thecompositions described herein (see Section 5.6.2) are used in thetreatment of a subject that has been infected by ExPEC.

Also provided herein are methods of inducing an immune response in asubject against ExPEC, comprising administering to the subject an E.coli O antigen described herein (see Section 5.2), a bioconjugatedescribed herein (see Section 5.4), or a composition described herein(see Section 5.6.2). In one embodiment, said subject has an ExPECinfection at the time of administration. In another embodiment, saidsubject does not have an ExPEC infection at the time of administration.

Also provided herein are methods of inducing the production ofopsonophagocytic antibodies against ExPEC in a subject, comprisingadministering to the subject an E. coli O antigen described herein (seeSection 5.2), a bioconjugate described herein (see Section 5.4), or acomposition described herein (see Section 5.6.2). In one embodiment,said subject has an ExPEC infection at the time of administration. Inanother embodiment, said subject does not have an ExPEC infection at thetime of administration.

In a specific embodiment, provided herein is a method for preventing anE. coli (e.g., ExPEC) infection in a subject, wherein said methodcomprises administering to a subject in need thereof an effective amountof a composition described in Section 5.6.2. The methods of preventingExPEC infection in a subject provided herein result in the induction ofan immune response in a subject comprising administering to the subjecta of a composition described in Section 5.6.2. One of skill in the artwill understand that the methods of inducing an immune response in asubject described herein result in vaccination of the subject againstinfection by the ExPEC strains whose O antigens are present in thecomposition(s).

In a specific embodiment, provided herein is a method for treating an E.coli (e.g., ExPEC) infection in a subject, wherein said method comprisesadministering to a subject in need thereof an effective amount of acomposition described in Section 5.6.2.

In certain embodiments, the immune response induced by an E. coli Oantigen described herein (see Section 5.2), a bioconjugate describedherein (see Section 5.4), or a composition described herein (see Section5.6.2) is effective to prevent and/or treat an ExPEC infection caused byany serotype, subserotype, or strain of ExPEC. In certain embodiments,the immune response induced by an E. coli O antigen described herein(see Section 5.2), a bioconjugate described herein (see Section 5.4), ora composition described herein (see Section 5.6.2) is effective toprevent and/or treat an ExPEC infection more than one serotype of ExPEC.

In a specific embodiment, the immune response induced by an E. coli Oantigen described herein (see Section 5.2), a bioconjugate describedherein (see Section 5.4), or a composition described herein (see Section5.6.2) is effective to prevent and/or treat an infection caused by E.coli of the O25 serotype. In a specific embodiment, said O25 serotype isO25B. In a specific embodiment, said O25 serotype is O25A.

In a specific embodiment, the immune response induced by an E. coli Oantigen described herein (see Section 5.2), a bioconjugate describedherein (see Section 5.4), or a composition described herein (see Section5.6.2) is effective to prevent and/or treat an infection caused by E.coli of the O1 serotype. In a specific embodiment, said O1 serotype isO1A. In another specific embodiment, said O1 serotype is O1B. In anotherspecific embodiment, said O1 serotype is O1C.

In a specific embodiment, the immune response induced by an E. coli Oantigen described herein (see Section 5.2), a bioconjugate describedherein (see Section 5.4), or a composition described herein (see Section5.6.2) is effective to prevent and/or treat an infection caused by E.coli of the O2 serotype.

In a specific embodiment, the immune response induced by an E. coli Oantigen described herein (see Section 5.2), a bioconjugate describedherein (see Section 5.4), or a composition described herein (see Section5.6.2) is effective to prevent and/or treat an infection caused by E.coli of the O6 serotype.

In a specific embodiment, the immune response induced by an E. coli Oantigen described herein (see Section 5.2), a bioconjugate describedherein (see Section 5.4), or a composition described herein (see Section5.6.2) is effective to prevent and/or treat an infection caused by twoor more of the following E. coli serotypes: O25 (e.g., O25B and O25A),O1 (e.g., O1A, O1B, and O1C), O2, and/or O6.

In a specific embodiment, the immune response induced by an E. coli Oantigen described herein (see Section 5.2), a bioconjugate describedherein (see Section 5.4), or a composition described herein (see Section5.6.2) is effective to prevent and/or treat an infection caused by eachof the following E. coli serotypes: O25 (e.g., O25B and O25A), O1 (e.g.,O1A, O1B, and O1C), O2, and 06.

In order to treat a subject having an ExPEC infection or immunize asubject against an ExPEC infection, the subject may be administered asingle composition described herein, wherein said composition comprisesone, two, three, four, or more E. coli antigens described herein. SeeSection 5.2. Alternatively, in order to treat a subject having an ExPECinfection or immunize a subject against an ExPEC infection, the subjectmay be administered multiple bioconjugates described herein, e.g., asubject may be administered two, three, four, or more bioconjugatesdescribed in Section 5.4. Alternatively, in order to treat a subjecthaving an ExPEC infection or immunize a subject against an ExPECinfection, the subject may be administered multiple compositionsdescribed herein, e.g., a subject may be administered two, three, four,or more compositions described in Section 5.6.2.

In certain embodiments, the immune response induced in a subjectfollowing administration of an E. coli O antigen described herein (seeSection 5.2), a bioconjugate described herein (see Section 5.4), or acomposition described herein (see Section 5.6.2) is effective to reducesymptoms resulting from an ExPEC infection. Symptoms of ExPEC infectionmay vary depending on the nature of the infection and may include, butare not limited to: dysuria, increased urinary frequency or urgency,pyuria, hematuria, back pain, pain while urinating, fever, chills,and/or nausea (e.g., in subjects having a urinary tract infection causedby ExPEC); high fever, headache, stiff neck, nausea, vomiting, seizures,sleepiness, and/or light sensitivity (e.g., in subjects havingmeningitis caused by ExPEC); fever, increased heart rate, increasedrespiratory rate, decreased urine output, decreased platelet count,abdominal pain, difficulty breathing, and/or abnormal heart function(e.g., in subjects having sepsis caused by ExPEC).

In certain embodiments, the immune response induced in a subjectfollowing administration of an E. coli O antigen described herein (seeSection 5.2), a bioconjugate described herein (see Section 5.4), or acomposition described herein (see Section 5.6.2) is effective to reducethe likelihood of hospitalization of a subject suffering from an ExPECinfection. In some embodiments, the immune response induced in a subjectfollowing administration of an E. coli O antigen described herein (seeSection 5.2), a bioconjugate described herein (see Section 5.4), or acomposition described herein (see Section 5.6.2) is effective to reducethe duration of hospitalization of a subject suffering from an ExPECinfection.

In another aspect, provided herein are methods of preventing and/ortreating an ExPEC infection in a subject caused by E. coli of the O25Bserotype by administering an antibody described herein, i.e., ananti-O25B antibody described herein. In particular embodiments, theneutralizing antibody is a monoclonal antibody.

5.7.1 Combination Therapies

In certain embodiments, an E. coli O antigen described herein (seeSection 5.2), a bioconjugate described herein (see Section 5.4), or acomposition described herein (see Section 5.6.2) is administered to asubject in combination with one or more other therapies (e.g.,antibacterial or immunomodulatory therapies). The one or more othertherapies may be beneficial in the treatment or prevention of an ExPECinfection or may ameliorate a symptom or condition associated with anExPEC infection. In some embodiments, the one or more other therapiesare pain relievers or anti-fever medications. In certain embodiments,the therapies are administered less than 5 minutes apart, less than 30minutes apart, 1 hour apart, at about 1 hour apart, at about 1 to about2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hoursto about 4 hours apart, at about 4 hours to about 5 hours apart, atabout 5 hours to about 6 hours apart, at about 6 hours to about 7 hoursapart, at about 7 hours to about 8 hours apart, at about 8 hours toabout 9 hours apart, at about 9 hours to about 10 hours apart, at about10 hours to about 11 hours apart, at about 11 hours to about 12 hoursapart, at about 12 hours to I8 hours apart, 18 hours to 24 hours apart,24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 72hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120hours part.

Any anti-bacterial agents known to one of skill in the art may be usedin combination with an E. coli O antigen described herein (see Section5.2), a bioconjugate described herein (see Section 5.4), or acomposition described herein (see Section 5.6.2). Non-limiting examplesof anti-bacterial agents include Amikacin, Amoxicillin,Amoxicillin-clavulanic acid, Amphothericin-B, Ampicillin,Ampicllin-sulbactam, Apramycin, Azithromycin, Aztreonam, Bacitracin,Benzylpenicillin, Caspofungin, Cefaclor, Cefadroxil, Cefalexin,Cefalothin, Cefazolin, Cefdinir, Cefepime, Cefixime, Cefmenoxime,Cefoperazone, Cefoperazone-sulbactam, Cefotaxime, Cefoxitin, Cefpirome,Cefpodoxime, Cefpodoxime-clavulanic acid, Cefpodoxime-sulbactam,Cefprozil, Cefquinome, Ceftazidime, Ceftibutin, Ceftiofur, Ceftobiprole,Ceftriaxon, Cefuroxime, Chloramphenicole, Florfenicole, Ciprofloxacin,Clarithromycin, Clinafloxacin, Clindamycin, Cloxacillin, Colistin,Cotrimoxazol (Trimthoprim/sulphamethoxazole), Dalbavancin,Dalfopristin/Quinopristin, Daptomycin, Dibekacin, Dicloxacillin,Doripenem, Doxycycline, Enrofloxacin, Ertapenem, Erythromycin,Flucloxacillin, Fluconazol, Flucytosin, Fosfomycin, Fusidic acid,Garenoxacin, Gatifloxacin, Gemifloxacin, Gentamicin, Imipenem,Itraconazole, Kanamycin, Ketoconazole, Levofloxacin, Lincomycin,Linezolid, Loracarbef, Mecillnam (amdinocillin), Meropenem,Metronidazole, Meziocillin, Mezlocillin-sulbactam, Minocycline,Moxifloxacin, Mupirocin, Nalidixic acid, Neomycin, Netilmicin,Nitrofurantoin, Norfloxacin, Ofloxacin, Oxacillin, Pefloxacin,Penicillin V, Piperacillin, Piperacillin-sulbactam,Piperacillin-tazobactam, Rifampicin, Roxythromycin, Sparfioxacin,Spectinomycin, Spiramycin, Streptomycin, Sulbactam, Sulfamethoxazole,Teicoplanin, Telavancin, Telithromycin, Temocillin, Tetracyklin,Ticarcillin, Ticarcillin-clavulanic acid, Tigecycline, Tobramycin,Trimethoprim, Trovafloxacin, Tylosin, Vancomycin, Virginiamycin, andVoriconazole.

In certain embodiments, a combination therapy comprises administrationof two or more E. coli O antigens described herein (see Section 5.2),bioconjugates described herein (see Section 5.4), and/or compositionsdescribed herein (see Section 5.6.2).

5.7.2 Patient Populations

In certain embodiments, an E. coli O antigen described herein (seeSection 5.2), a bioconjugate described herein (see Section 5.4), or acomposition described herein (see Section 5.6.2) is administered to anaïve subject, i.e., a subject that does not have an ExPEC infection orhas not previously had an ExPEC infection. In one embodiment, an E. coliO antigen described herein (see Section 5.2), a bioconjugate describedherein (see Section 5.4), or a composition described herein (see Section5.6.2) is administered to a naïve subject that is at risk of acquiringan ExPEC infection.

In certain embodiments, an E. coli O antigen described herein (seeSection 5.2), a bioconjugate described herein (see Section 5.4), or acomposition described herein (see Section 5.6.2) is administered to asubject who has been diagnosed with an ExPEC infection. In someembodiments, an E. coli O antigen described herein (see Section 5.2), abioconjugate described herein (see Section 5.4), or a compositiondescribed herein (see Section 5.6.2) is administered to a subjectinfected with ExPEC before symptoms manifest or symptoms become severe(e.g., before the patient requires hospitalization).

In certain embodiments, an E. coli O antigen described herein (seeSection 5.2), a bioconjugate described herein (see Section 5.4), or acomposition described herein (see Section 5.6.2) is administered to asubject who has been diagnosed with an UPEC infection. In someembodiments, an E. coli O antigen described herein (see Section 5.2), abioconjugate described herein (see Section 5.4), or a compositiondescribed herein (see Section 5.6.2) is administered to a subjectsuffering from reoccurring urinary tract infections. In someembodiments, an E. coli O antigen described herein (see Section 5.2), abioconjugate described herein (see Section 5.4), or a compositiondescribed herein (see Section 5.6.2) is administered to a subjectsuffering from reoccurring urinary tract infections, but is healthy atthe moment of treatment. In some embodiments, an E. coli O antigendescribed herein (see Section 5.2), a bioconjugate described herein (seeSection 5.4), or a composition described herein (see Section 5.6.2) isadministered to a subject having or at risk of acquiring bacteremia orsepsis.

In some embodiments, a subject to be administered an E. coli O antigendescribed herein (see Section 5.2), a bioconjugate described herein (seeSection 5.4), or a composition described herein (see Section 5.6.2) isan animal. In certain embodiments, the animal is a bird. In certainembodiments, the animal is a canine. In certain embodiments, the animalis a feline. In certain embodiments, the animal is a horse. In certainembodiments, the animal is a cow. In certain embodiments, the animal isa mammal, e.g., a horse, swine, mouse, or primate. In a specificembodiment, the subject is a human.

In certain embodiments, a subject to be administered an E. coli Oantigen described herein (see Section 5.2), a bioconjugate describedherein (see Section 5.4), or a composition described herein (see Section5.6.2) is a human adult. In certain embodiments, a subject to beadministered an E. coli O antigen described herein (see Section 5.2), abioconjugate described herein (see Section 5.4), or a compositiondescribed herein (see Section 5.6.2) is a human adult more than 50 yearsold. In certain embodiments, a subject to be administered an E. coli Oantigen described herein (see Section 5.2), a bioconjugate describedherein (see Section 5.4), or a composition described herein (see Section5.6.2) is an elderly human subject.

In certain embodiments, a subject to be administered an E. coli Oantigen described herein (see Section 5.2), a bioconjugate describedherein (see Section 5.4), or a composition described herein (see Section5.6.2) is a human child. In certain embodiments, a subject to beadministered an E. coli O antigen described herein (see Section 5.2), abioconjugate described herein (see Section 5.4), or a compositiondescribed herein (see Section 5.6.2) is a human infant.

In certain embodiments, a subject to be administered an E. coli Oantigen described herein (see Section 5.2), a bioconjugate describedherein (see Section 5.4), or a composition described herein (see Section5.6.2) is a premature human infant. In some embodiments, a subject to beadministered an E. coli O antigen described herein (see Section 5.2), abioconjugate described herein (see Section 5.4), or a compositiondescribed herein (see Section 5.6.2) is a human toddler. In certainembodiments, a subject to be administered an E. coli O antigen describedherein (see Section 5.2), a bioconjugate described herein (see Section5.4), or a composition described herein (see Section 5.6.2) isadministered is not an infant of less than 6 months old.

In certain embodiments, a subject to be administered an E. coli Oantigen described herein (see Section 5.2), a bioconjugate describedherein (see Section 5.4), or a composition described herein (see Section5.6.2) is an individual who is pregnant. In certain embodiments, asubject to be administered an E. coli O antigen described herein (seeSection 5.2), a bioconjugate described herein (see Section 5.4), or acomposition described herein (see Section 5.6.2) is a woman who hasgiven birth 1, 2, 3, 4, 5, 6, 7, or 8 weeks earlier.

In certain embodiments, a subject to be administered an E. coli Oantigen described herein (see Section 5.2), a bioconjugate describedherein (see Section 5.4), or a composition described herein (see Section5.6.2) is an individual at increased risk of ExPEC (e.g., animmunocompromised or immunodeficient individual). In certainembodiments, a subject to be administered an E. coli O antigen describedherein (see Section 5.2), a bioconjugate described herein (see Section5.4), or a composition described herein (see Section 5.6.2) is anindividual in close contact with an individual having or at increasedrisk of ExPEC infection.

In certain embodiments, a subject to be administered an E. coli Oantigen described herein (see Section 5.2), a bioconjugate describedherein (see Section 5.4), or a composition described herein (see Section5.6.2) is a health care worker (e.g., a doctor or nurse). In certainembodiments, a subject to be administered an E. coli O antigen describedherein (see Section 5.2), a bioconjugate described herein (see Section5.4), or a composition described herein (see Section 5.6.2) isimmunocompromised (e.g., suffers from HIV infection) orimmunosuppressed.

In certain embodiments, a subject to be administered an E. coli Oantigen described herein (see Section 5.2), a bioconjugate describedherein (see Section 5.4), or a composition described herein (see Section5.6.2) has diabetes. In certain embodiments, a subject to beadministered an E. coli O antigen described herein (see Section 5.2), abioconjugate described herein (see Section 5.4), or a compositiondescribed herein (see Section 5.6.2) has multiple sclerosis.

In certain embodiments, a subject to be administered an E. coli Oantigen described herein (see Section 5.2), a bioconjugate describedherein (see Section 5.4), or a composition described herein (see Section5.6.2) has a condition that requires them to use a catheter. In certainembodiments, a subject to be administered an E. coli O antigen describedherein (see Section 5.2), a bioconjugate described herein (see Section5.4), or a composition described herein (see Section 5.6.2) has a spinalcord injury.

5.7.3 Dosage and Frequency of Administration

The amount of an E. coli O antigen described herein (see Section 5.2), abioconjugate described herein (see Section 5.4), or a compositiondescribed herein (see Section 5.6.2) which will be effective in thetreatment and/or prevention of an ExPEC infection will depend on thenature of the disease, and can be determined by standard clinicaltechniques. Administration of the O-antigen, bioconjugate and/orcomposition can be done via various routes known to the clinician, forinstance subcutaneous, parenteral, intravenous, intramuscular, topical,oral, intradermal, transdermal, intranasal, etc. In one embodiment,administration is via intramuscular injection.

The precise dosage to be employed in the formulation will also depend onthe route of administration, and the seriousness of the infection, andshould be decided according to the judgment of the practitioner and eachsubject's circumstances. For example, effective dosages may also varydepending upon means of administration, target site, physiological stateof the patient (including age, body weight, health), whether the patientis human or an animal, other medications administered, and whethertreatment is prophylactic or therapeutic. Treatment dosages areoptimally titrated to optimize safety and efficacy.

In certain embodiments, an in vitro assay is employed to help identifyoptimal dosage ranges. See Section 5.8. Effective doses may beextrapolated from dosage response curves derived from in vitro or animalmodel test systems.

In certain embodiments, exemplary dosages for glycoconjugate basedvaccines (e.g., compositions comprising bioconjugates) range from about0.1 μg to 400 μg of carbohydrate per dose. In other embodiments,exemplary dosages for glycoconjugate based vaccines (e.g., compositionscomprising bioconjugates) range from about 0.1 μg to 4000 μg ofprotein(s) per dose. In certain embodiments, an exemplary dosage for aglycoconjugate based vaccine (e.g., a composition comprisingbioconjugates) comprises 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, or 50 gig of carbohydrate(s) per dose. In certain embodiments,an exemplary dosage for a glycoconjugate based vaccine (e.g., acomposition comprising bioconjugates) comprises 50, 55, 60, 65, 70, 75,80, 85, 90, 95, or 100 μg of protein(s) per dose. In certain exemplaryembodiments, a dosage for administration to a human corresponds to 0.5ml containing about 1-10, e.g. about 2-6, e.g. about 4 jpg ofpolysaccharide for each of the glycoconjugates included.

In certain embodiments, an E. coli O antigen described herein (seeSection 5.2), a bioconjugate described herein (see Section 5.4), or acomposition described herein (see Section 5.6.2) is administered to asubject once as a single dose. In certain embodiments, an E. coli Oantigen described herein (see Section 5.2), a bioconjugate describedherein (see Section 5.4), or a composition described herein (see Section5.6.2) is administered to a subject as a single dose followed by asecond dose 3 to 6 weeks later. In accordance with these embodiments,booster inoculations may be administered to the subject at 6 to 12 monthintervals following the second inoculation. In certain embodiments, thebooster inoculations may utilize a different E. coli O antigen,bioconjugate, or composition. In some embodiments, the administration ofthe same E. coli O antigen, bioconjugate, or composition may be repeatedand the administrations may be separated by at least 1 day, 2 days, 3days, 5 days, 7 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75days, 3 months, or at least 6 months. In certain embodiments, an E. coliO antigen described herein (see Section 5.2), a bioconjugate describedherein (see Section 5.4), or a composition described herein (see Section5.6.2) is administered to a subject as a single dose once per year.

In certain embodiments, an E. coli O antigen described herein (seeSection 5.2), a bioconjugate described herein (see Section 5.4), or acomposition described herein (see Section 5.6.2) is administered to asubject as 2, 3, 4, 5 or more doses 2 weeks, 3 weeks, 4 weeks, 5 weeksor 6 weeks apart. In some embodiments, 2, 3, 4, 5 or more doses of an E.coli O antigen described herein (see Section 5.2), a bioconjugatedescribed herein (see Section 5.4), or a composition described herein(see Section 5.6.2) are administered to a subject 2, 3, 4, 5 or 6 weeksapart at a dosage of 0.1 μg to 0.5 mg, 0.1 μg to 0.4 mg, 0.1 μg to 0.3mg, 0.1 μg to 0.2 mg, or 0.1 μg to 0.1 mg carbohydrate content. Incertain embodiments, the E. coli O antigen, bioconjugate, or compositionadministered is the same each time. In certain embodiments, the E. coliO antigen, bioconjugate, or composition administered is different eachtime.

For passive immunization with an antibody (e.g., an anti-O25B antibody),the dosage can range from about 0.0001 to 100 mg of antibody per kg ofbody weight, or from 0.01 to 5 antibody per kg of body weight. Forexample, dosages can be 1 mg/kg body weight or 10 mg/kg body weight orwithin the range of 1-10 mg/kg or in other words, 70 mg or 700 mg orwithin the range of 70-700 mg, respectively, for a 70 kg patient. Anexemplary treatment regime entails administration once per every twoweeks or once a month or once every 3 to 6 months for a period of oneyear or over several years, or over several year-intervals. Intervalscan be irregular and altered based on blood levels of antibody in thepatient.

5.8 Assays

Assay for Assessing Ability of Bioconjugates to Induce an ImmuneResponse

The ability of the bioconjugates/compositions described herein togenerate an immune response in a subject can be assessed using anyapproach known to those of skill in the art or described herein. In someembodiments, the ability of a bioconjugate to generate an immuneresponse in a subject can be assessed by immunizing a subject (e.g., amouse) or set of subjects with a bioconjugate described herein andimmunizing an additional subject (e.g., a mouse) or set of subjects witha control (PBS). The subjects or set of subjects can subsequently bechallenged with ExPEC and the ability of the ExPEC to cause disease(e.g., UTI) in the subjects or set of subjects can be determined. Thoseskilled in the art will recognize that if the subject or set of subjectsimmunized with the control suffer(s) from disease subsequent tochallenge with the ExPEC but the subject or set of subjects immunizedwith a bioconjugate(s) or composition thereof described herein sufferless from or do not suffer from disease, then the bioconjugate is ableto generate an immune response in a subject. The ability of abioconjugate(s) or composition thereof described herein to induceantiserum that cross-reacts with an O antigen from ExPEC can be testedby, e.g., an immunoassay, such as an ELISA.

In Vitro Bactericidal Assays

The ability of the bioconjugates described herein to generate an immuneresponse in a subject can be assessed using a serum bactericidal assay(SBA) or opsonophagocytotic killing assay (OPK), which represents anestablished and accepted method that has been used to obtain approval ofglycoconjugate-based vaccines. Such assays are well-known in the artand, briefly, comprise the steps of generating and isolating antibodiesagainst a target of interest (e.g., an O antigen, e.g., O25B, of E.coli) by administering to a subject (e.g., a mouse) a compound thatelicits such antibodies. Subsequently, the bactericidal capacity of theantibodies can be assessed by, e.g., culturing the bacteria in question(e.g., E. coli of the relevant serotype) in the presence of saidantibodies and complement and—depending on the assay—neutrophilic cellsand assaying the ability of the antibodies to kill and/or neutralize thebacteria, e.g., using standard microbiological approaches.

5.9 Kits

Provided herein is a pharmaceutical pack or kit comprising one or morecontainers filled with one or more of the ingredients of thecompositions described herein (see Section 5.6.2), such as one or moreE. coli antigens (see Section 5.2) and/or bioconjugates (see Section5.4) provided herein. Optionally associated with such container(s) canbe a notice in the form prescribed by a governmental agency regulatingthe manufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration. The kits encompassed herein can be used in theabove methods of treatment and immunization of subjects.

6. EXAMPLES Methods

Agglutination

A process in which cells or lysed cell mass is mixed with antiserumcontaining antibodies specific for a polymeric structure, e.g. Oantigen. Visible, insoluble aggregates form when the antiserumrecognizes the cellular structures. This method is classically used toidentify O, K, and H serotypes. See DebRoy, et al., (2011) Animal healthresearch reviews/Conference of Research Workers in Animal Diseases 12,169-185

LPS Sample Preparation for Analysis by SDS PAGE

LPS of Gram-negative cells is composed of a lipid A base, modified witha core oligosaccharide providing the attachment for the O antigen. Toanalyze the LPS of clinical isolates, cells were grown in standard LBmedium at 37° C. for 24 h, and biomass corresponding to 1 ml of culturewith an OD600 of 2 was collected and lysed in 1× Lämmli sample bufferand incubated at 95° C. for 10 minutes. Extracts were further treatedfor 1 hour at 65° C. to remove any protein signal using 1 g/l proteinaseK. The treated extracts were separated by SDS PAGE and LPS wasvisualized by silver staining or Western blotting using appropristeantiserum.

LPS Preparation for Coating of ELISA Plates

LPS was prepared using a method described by Apicella, (2008) MethodsMol Biol 431, 3-13, and further purified as described by Perdomo andMontero, (2006) Biotecnologfa Aplicada 23:124-129.

2AB OPS HPLC: ‘LLO Fingerprinting’

This method is used to analyze the structure of UPP linked OPS.

To extract UPP-linked glycans, E. coli cells were washed with 0.9% NaCland lyophilized. The dried cells were extracted with organic solvent(Methanol:Water (M:W=17:3 to 19:1, v/v), and/orChloroform:Methanol:Water mixtures of optimized ratios (e.g.C:M:W=10:10:3; v/v/v)). The extracts were dried under a stream of N2,and resuspended in C:M:W=3:48:47. To purify the extracted glycolipids,the 3:48:47 resuspension was passed through a tC₁₈ Sep-PAK cartridge.The cartridge was conditioned with 10 ml methanol, followed byequilibration with 10 mil 3:48:47 (C:M:W). After loading of the sample,the cartridge was washed with 10 ml 3:48:47 (C:M:W) and eluted with 5 mlmethanol and 5 ml 10:10:3 (C:M:W). The combined elutions were driedunder N₂. The glycolipid samples were hydrolyzed by dissolving the driedsamples in 2 ml n-propanol:2 M trifluoroacetic acid (1:1), heating to50° C. for 15 min, and then evaporating to dryness under N₂ (Glover, etal., Proc Natl Acad Sci USA 102(40): 14255-9). The dried samples wereonce more resuspended in 3:48:47 and passed through a tC₁₈ cartridge,and the flow through was dried under N2. Labeling with 2-AB and glycancleanup was performed using the paper disk method as described (Bigge,et al., Anal Biochem 230(2): 229-38; Merry, et al., Anal Biochem 304(1):91-9).

2-AB labeled glycans were separated by HPLC using a GlycoSep-N normalphase column according to Royle et al. but modified to a three solventsystem (Royle, et al., Anal Biochem 304(1): 70-90). Solvent A was 10 mMammonium formate pH 4.4 in 80% acetonitrile. Solvent B was 30 mMammonium formate pH 4.4 in 40% acetonitrile. Solvent C was 0.5% formicacid. The column temperature was 30° C. and 2-AB labelled glycans weredetected by fluorescence (excitation λex=330 nm, emission λem=420 nm).Gradient conditions were a linear gradient of 100% A to 100% B over 160min at a flow rate of 0.4 ml/min, followed by 2 min 100% B to 100% C,increasing the flow rate to 1 ml/min. The column was washed for 5 minwith 100% C, returning to 100% A over 2 min and running for 15 min at100% A at a flow rate of 1 ml/min, then returning the flow rate to 0.4ml/min for 5 min. Samples were injected in water.

Deacetylation Assay:

An equivalent of 2-AB labeled glycan is dried in at 30° C., resuspendedin 50 μl water with (sample) or without (mock) 200 mM NaOH (pH=14), andincubated for 25 hours at 37° C. The solution is then brought to roomtemperature and neutralized by the addition of 200 mM HCl solution(pH≈1). After drying in the speed vacuum at 30° C., the sample isrelabeled with 2AB and analysis by HPLC.

Hydrazinolysis HPLC

The same normal phase HPLC technique described above was used toseparate OPS released from bioconjugates after hydrazinolysis. Prior tohydrazinolysis, bioconjugates corresponding to 1 mg protein werecompletely dried under a stream of N₂. Polysaccharide release wasperformed using the Ludger Liberate Hydrazinolysis Glycan Release kit(Ludger # LL-HYDRAZ-A2) according to the manufacturer's instructions.Briefly, 450 μl hydrazine were added to the dried samples under ablanket of N2 and incubated for 16 hours at 85° C. The hydrazine wasremoved by evaporation under N2 at 45° C. Re-N-acetylation of thepolysaccharides was performed by incubation in 471 μl 4.5% aceticanhydride in 1 M sodium bicarbonate for two hours on ice. Thereafter,600 μl of a 5% TFA solution was added and the samples were hydrolyzedfor another hour on ice. Purification was performed on an EB20 columnusing the corresponding buffers EB20 A and B.

The released and purified polysaccharides were labeled with 2-AB andanalyzed by NP-HPLC like described for the LLO samples. Peaks ofinterest were collected and identified by MS/MS.

MS and MS/MS of HPLC Peaks

To analyze the monosaccharide sequence of an OPS molecule of interest,mass spectroscopic analysis was performed. Dried, collected fractionscorresponding to specific HPLC peaks were resuspended in 5 ul 10%acetonitrile (ACN), 0.1% trifluoro acetic acid (TFA) and mixed 1:1 withmatrix solution (40 mg/ml DHB in 50% ACN, 0.1% TFA) on the target plate.MS and MS/MS data were manually acquired in the positive ion mode on anUltraflex-II MALDI-ToF/ToF mass spectrometer (Bruker Daltonik GmbH,Bremen, Germany). MS/MS were obtained using the LIFT method. A standardpeptide mixture (Bruker Daltonik GmbH) was used for externalcalibration. Spectra were exported using the Flex Analysis software(Bruker Daltonik GmbH) and manually analyzed.

Host Cells

Bioconjugates were produced by recombinant E. coli cells expressing, viaplasmid(s), carrier protein(s) and the oligosaccharyl transferase fromC. jejuni (PglB), and OPS from cosmids or chromosomal insertion mutants.

Genetically detoxified EPA (Exotoxin A from Pseudomonas aeruginosacontaining the mutations L552V, ΔE553) was used as a carrier protein,and was modified to comprise 2 or 4 glycosylation sites (referred toherein as 2S-EPA and 4S-EPA, respectively) and a C-terminal HIS Tag, andwas expressed from a pBR322 derived, arabinose inducible plasmid (seeIhssen, et al., (2010) Microbial cell factories 9, 61).

MBP (Maltose binding protein), a native periplasmic, soluble E. coliprotein, was expressed from a pGVXN579. pGVXN579 is a modified pMAL-p2X(New England Biolabs) plasmid encoding three bacterial N glycosylationconsensus sequences in a row followed by a Myc-Tag C-terminally fused tothe maltose binding protein ORF encoded on the plasmid. This setupallowed affinity purification of the MBP bioconjugate independent of aHIS-tag. Induction of expression is controlled by the tac promoter andinducible using IPTG.

The PglB protein was expressed from plasmid pEXT21 (an EcoRI/BamHIfragment from pMAF10 (Feldman et al., 2005, PNAS USA 102(8):3016-3021)was cloned into pEXT21 with a C-terminally fused HA-tag. Variants of theexpression plasmid are codon optimization (pGVXN939), codon optimizationwith a deletion of the glycosylation site (pGVXN948), and removedHA-Ttag (pGVXN970) and codon optimization and deleted HA tag (pGVXN971).

Clinical isolates were analyzed for their capability to synthetize acertain OPS using agglutination, Western blot, silver staining, LLOfingerprinting, PCR serotyping, or similar technologies that allowidentification of the OPS structural characteristics, and also for theirantibiotic resistance phenotype. Certain clinical isolates were furtherchromosomally deleted for the ligase enzyme, WaaL, for enhancing OPSavailability for protein glycosylation or OPS analysis.

To further analyze clinical isolates, the rfb cluster of the laboratorystrain W3110 was replaced by the rfb cluster cloned from clinicalisolates, and OPS biosynthesis was analyzed. The waaL gene was deletedto enhance efficiency of bioconjugate production.

Cluster exchanges and waaL deletions were achieved by homologousrecombination using an optimized method (see International Patentapplication No. PCT/EP2013/068737) or published procedures (Datsenko andWanner, (2000) Proc Natl Acad Sci USA 97, 6640-6645). For OPS clusterexchange, the rfb cluster of interest of a clinical isolate was clonedinto the counter selection plasmid pDOC-C, along with an antibioticresistance cassette, for subsequent integration into the rjb locus of E.coli strain W3110 (Kuhlman and Cox, 2010, Nucleic acids research 38,e92; Lee et al, 2009, BMC Microbiol 9, 252). Homologous recombination oflarge rfb clusters of interest was achieved by using DNA flanking therfb cluster coding sequence of W3110 of 0.5 to 1.5 kilobases in lengthand in vivo linearization of the insert DNA from plasmid borne rfb. Theresulting strain contained a replaced rb cluster (with and withoutantibiotic resistance cassette), i.e. the rb cluster of W3110 wasreplaced for a DNA molecule between the gale and gnd genes by theanalogous stretch isolated from a clinical E. coli isolate.

In certain experiments, W3110 strains containing a cosmid encoding therfb cluster of a given E. coli serotype were used as host strains.

For the production of recombinantly expressed bioconjugates in W3110based strains, W3110 borne genes that interfere with recombinant OPSproduction were deleted. For example, for the production host cell ofO25B bioconjugates, the gtrABS cluster of W3110 was deleted.

To achieve this, homologous recombination according to a publishedmethod (Bloor A E, Cranenburgh R M. Appl Environ Microbiol. 2006 April;72(4):2520-5.) using homology sequences flanking upstream the gtrA geneand downstream of the gtrS gene.

To assemble production strains, the host strain was transformed with apglB and a carrier expression plasmid by transformation. See Wacker etal., 2002, Science 298:1790-1793.

Bioconjugate Production

Bioconjugate production was performed by growing host cells andpurifying bioconjugates produced in the periplasmic space. Growth wasperformed either in shake flasks or in an industrial scale fed batchfermentation process.

Shake flask cultivations were performed at 37° C., using a mediumcomposed of the appropriate antibiotics in terrific broth sometimessupplemented with 5 mM MgCl2. Medium of was inoculated at an OD of 0.05with an overnight culture from freshly transformed production cells,grown until mid-log phase, induced with 0.2% arabinose and 1 mM IPTG,further grown and harvested after 20 hs of growth.

Fed Batch Fermentations

An aliquot of a production cell line bank was used to inoculate a shakeflask containing Soy LB medium with the appropriate antibiotics. Theshake flask was incubated at 180 rpm, 37° C. for approximately 12 hours.The batch media without complements were sterilized directly inside thebioreactor (33 min at >121° C.), cooled, and complements were added. 4 MKOH or 25% phosphoric acid were attached to the fermenter for pHregulation and pH was adjusted to pH7. Inoculation of the bioreactor andbatch culture from the pre-culture was done to yield an initial OD600 of0.005. pH was stably maintained by the addition of 4 M KOH or 25%phosphoric acid. Dissolved oxygen tension (DO) is maintained. Overheadpressure was maintained at 600 mbar. Product formation was induced withL-Arabinose (0.1%) and/or IPTG (1 mM). Immediately after induction, feedwas initiated by addition of feed medium containing 2.5% Arabinose andIPTG. 24±2 hours after induction, the bioreactor was cooled to 25° C.,feed was stopped and harvesting was performed by tangential flowfiltration or centrifugation.

The biomass was lysed in 0.5% TRITON™ X-100 by disruption during 4cycles of high pressure homogenization at 800 bar.

Bioconjugates were purified by column chromatography. Variouschromatographic techniques were used to prepare bioconjugates, mainlyIMAC, Q-resin based anionic exchange chromatography (AEC), and sizeexclusion chromatography (SEC). See, e.g., Saraswat et al., 2013,Biomed. Res. Int. ID #312709 (p. 1-18) and WO 2009/104074 fordescription of such methods.

Bioconjugate Production for Preclinical Experiments

From the pre-culture, a defined amount was transferred to a bioreactorcontaining a rich media at 35±0.2° C. The pH and dissolved oxygentension were maintained. Agitation rate reached 700 rpm.

When cell density reached OD₆₀₀=40±5, product formation was induced withL-Arabinose (0.1%) and IPTG (1 mM). Feed was initiated 24±2 hours afterinduction and the bioreactor was cooled. As soon as the temperaturereached 25° C., feed was stopped and the cells were collected.

High Pressure Homogenization

A biomass corresponding to 50 L at harvest was thawed for 1 day at 2 to80C. Then 2.5 L of Lysis and Clarification Buffer was added to thecontainer. TRITON™ X-100 was added to a final concentration of 0.5% andthe completely thawed cells were disrupted by 4 cycles of high pressurehomogenization at 800 bar. Cells were harvested and washed usingstandard techniques.

Monosaccharide Composition Analysis:

Bioconjugates containing approximately 8 ug polysaccharide werehydrolyzed for six hours in 104 μl 3 M TFA at 99° C. TFA was removed byevaporation and samples were washed once with 500 μl 2-propanol. Theresulting monosaccharides were suspended in 100 μl labeling mixcontaining 87.1 mg/ml 1-phenyl-3-methyl-5-pyrazolone (PMP), 50% MeOH and150 mM NaOH. Labeling was performed during 60 minutes at 70° C. Thesamples were neutralized by the addition of 50 μl 300 mM HCl and 20 μl100 mM Tris/HCl pH 7.0. The PMP-labeled monosaccharides were purified byextraction, once with 1 ml di-buthyl ether and three times with 1 mlCHCl₃.

The PMP derivatized monosaccharides were separated by RP-HPLC(Merck-Hitachi) on a C18 Inertsil ODS-3 column (GL Sciences) equippedwith a pre-column. A two-step gradient from 100% buffer A (13%acetonitrile, 87% H2O (0.045% KH2PO4, 0.05% triethylamine, pH 7.0) to50% buffer A/50% buffer B (21% acetonitrile, 79% H2O (0.045% KH2PO4,0.05% triethylamine, pH 7.0) over 4 minutes to 100% buffer B over 47minutes was applied at 35° C. and a flow rate of 1 ml/min. The injectionvolume was 50 μl and elution was monitored by online UV-detection at 250nm. The individual peaks were identified by overlay with chromatogramsof the commercially available monosaccharide standards D-glucose(Sigma-Aldrich # G7528), L-rhamnose (Sigma-Aldrich # R3875),N-acetyl-D-glucosamine (Sigma-Aldrich # A8625) and N-acetyl-L-fucosamine(Omicron Biochemicals # FUC-006).

Example 1: Epidemiology

To determine the serotype distribution of urinary tract infection(UTI)-causing E. coli, an epidemiology study was performed. Over 1800 E.coli isolates form human urine samples were collected from subjects inSwitzerland and the O antigen serotypes (OPS) from each sample wasanalyzed using classical agglutination techniques. See FIG. 4

Isolated human urine samples were analyzed to determine the identity ofpathogens therein and their antibiotic resistance patterns. E. coliisolates were obtained from the samples following the analysis. E. coliisolates were identified by classical microbiological exclusion andinclusion strategies involving growth on chrome (CPS3) and MacConkeyagar. E. coli isolates further were analyzed using an agglutinationassay to determine their O antigen serotype. See DebRoy et al. (2011)Animal health research reviews/Conference of Research Workers in AnimalDiseases 12, 169-185. Isolates from the same O antigen serogroups werefurther analyzed to determine the chemical structure of the O chain fromeach isolate. See Table 1A. Certain isolated E. coli strains weredetermined to be antibiotic resistant, including identification offluoroquinolone-resistant strains and extended-spectrum beta-lactamase(ESBL) producing strains.

TABLE 1A Distribution of the most common UTI-associated E. coliserotypes from a collection of 1841 urine samples collected inSwitzerland in 2012. Shown is the serotype distribution of samples froma relevant subpopulation of 671 subjects, and the distribution fromall** samples. Most prevalent E. coli serotypes associated with UTICommunity and Community acquired hospital acquired UTI in 18-70 yearsUTI in all ages** O-serotype old* (n = 671) O-serotype (n = 1871) 610.75% 2 8.75% 2 9.55% 6 8.47% 25 6.87% 25 8.37% 1 5.52% 75 4.56% 45.37% 1 4.29% 75 4.78% 8 3.86% 8 3.43% 18 3.53% 18 3.28% 4 3.26% 153.28% 15 2.39% 73 2.24% 73 2.17% 16 2.24% 16 1.85% 7 1.94% 7 1.68%

Serotypes O1, O2, O4, O6, O7, O8, O16, O18, O25, O73, and O75 wereisolated from subjects independent of location, time of isolation,symptoms, and target population, suggesting these to be the predominantserotypes of uropathogenic E. coli (UPEC). Accordingly, theidentification of the most prevalent O antigen serotypes indicates thatO-antigen specific vaccines could be limited to a subset of serotypes,namely those most associated with disease, as identified in the studydescribed in this example.

A retrospective analysis of UTI serotypes in 1323 isolates from the pastthree decades in the US obtained from the E. coli Reference center(ECRC) allowed a thorough comparison to literature and the current datafrom Switzerland. The prevalence of the top 20 serotypes was foundindependently on location, time of isolation, symptoms, or targetpopulation and suggests predominant serotypes associated to UPEC (seeTable 1B).

TABLE 1B Prevalence of most common UTI associated serotypes fromselected literature ranging from 1987-2011 and from retrospectivelyanalysed US data from 2000-2011 (ECRC). INDICATION US 2000-2010 TOTALPYELO- 315 (all UTI UTI CYSTITIS NEPHRITIS specimen except availableavailable available fecal, all ages, data data data F + M) Number offrom 1860 from 1089 from 373 non-typable were Serotype isolates isolatesisolates not available! O1 4.8% 4.1% 5.4% 7.0% O2 7.1% 4.9% 15.3% 14.0%O4 7.8% 6.0% 3.2% 3.2% O6 16.9% 16.3% 7.8% 18.7% O7 3.3% 2.4% 2.4% 1.9%O8 1.7% 3.2% 0.8% 3.5% O15 0.6% 1.5% 0.8% 1.3% O16 4.3% 3.2% 7.2% 1.9%O18 7.0% 7.1% 6.7% 7.0% O21 na na na 1.3% O22 0.6% 0.6% 0.5% 0.0% O253.0% 4.8% 0.5% 8.6% O75 7.5% 6.0% 8.6% 3.8% O83 1.9% 0.7% 0.5% 1.3% O201.6% O77 2.2% O82 1.9% others and 33.3% 39.2% 40.2% non typable/ notavailable other O- 21.0% types (NT not available)Isolates from serotypes described were calculated as percentage on thetotal number of isolates (Andreu et al., 1997, J Infect Dis 176:464-469;Blanco et al., 1996, Eur J Epidemiol 12:191-198; Fathollahi et al.,2009, Iranian Journal of Clinical Infectious Diseases 4:77-81; Johnsonet al., 2005, J Clin Microbiol 43:6064-6072; Molina-Lopez et al., 2011,Journal of infection in developing countries 5:840-849; Sandberg et al.,1988, J Clin Microbiol 26:1471-1476; K. L. 2007, The Journal ofinfection 55:8-18; Terai et al., 1997, Int J Urol 4:289-294.) In certaincases specific data was not available; therefore the percentage numberscan only give an indication on the overall serotype distribution fromdifferent UTI isolates in described studies and should be consideredwith caution. The other described serotypes identified however lessprevalent (O15, O20, O21, O22, O77 and O82) also are included.All information from epidemiology analysis taken together, the 10predominant serotypes could cover an estimated 60-80% of E. coliinfections, assuming coverage of subportions of the non typeablestrains. Furthermore, the data shows the unexpected importance of theO25 serotype in the epidemiology study from Switzerland, when comparedto literature data and recent data from the USA. See Tables 1A and B.

O antigen serotypes of E. coli often are composed of subtypes, which aredistinct, yet structurally and antigenically similar. To identifyunknown/unreported subtypes among the collected clinical isolates, andto identify the most prevalent O antigen subtypes, the chemicalstructures of the O antigens from the most prevalent serotypes wereanalyzed in more detail.

Example 2: E. coli O25

In recent years, increased occurrence of O25-positive strains has beenobserved (see George and Manges (2010) Epidemiol Infect 138, 1679-1690)and is evidenced by the study described in Example 1, where the O25serotype was found to be one of the top four E. coli serotypes in termsof prevalence.

O25A

An O antigen repeat unit structure of the E. coli O25 serotype has beenpublished previously (see Kenne et al., 1983, Carbohydrate Research 122,249-256; and Fundin et al., 2003, Magnetic Resonance in Chemistry 41, 4)and is presented in FIG. 2B. An rfb cluster related to the O25 O antigenfrom E. coli strain E47a is publicly available (GenBank GU014554), andis presented in FIG. 2A. E. coli E47a is used as a reference strain forO25 serotyping. Further rfb cluster sequence information is availablefrom the genome sequence of a strain causing asymptomatic bacteriuria,E. coli 83972. (see Zdziarski et al., 2010, PLoS Pathog 6, e1001078).Although phenotypic O25 expression has not been confirmed, rfb clustersequences of E. coli E47a and 83972 are 99.49% identical, stronglysuggesting they encode the same O antigen.

The O antigen from E. coli strains 83972 and E47a is designated hereinas “O25A,” because, as described below, a novel E. coli O antigen,designated “O25B,” was identified based on analysis of the clinicalisolates obtained in the epidemiology study described in Example 1,above.

The functionalities for the predicted gene products of E. coli strains83972 and E47a have been proposed. See Table 2; GenBank GU014554; andSzijarto, et al. (2012) FEMS Microbiol Lett 332, 131-136.

TABLE 2 O25A O antigen gene predictions from the rfb cluster aspublished by Wang, et al. (2010) J Clin Microbiol 48, 2066-2074; seealso GenBank GU014554. Most meaningful Gene homology/Protein[organism],name Putative Function accession, max. identity (BLAST) rmlBdTDP-Glucose 4,6- dTDP-Glucose 4,6-dehydratase dehydratase (E. coliIAI39), YP_002406996.1, 98% rmlD dTDP-6-Deoxy-D-glucosedTDP-6-Deoxy-L-mannosedehydrogenase 3,5-epimerase (E. coli), ACA24825.1,97% rmlA Glucose-1-phosphate Glucose-1-phosphate thymidylyltransferasethymidylyltransferase (E. coli IAI39), YP_002406998.1, 99% rmlCdTDP-4-dehydrorhamnose RmlC (E. coli), ACA24796.1, 70% 3,5-epimerase WzxO antigen flippase O-antigen transporter (E. coli), WP_000021239.1, 100%wekA Glycosyltransferase dTDP-Rha: Glc-Rha(Ac)-GlcNAc-UPP α-1,3-rhamnosyltransferase (E. coli), WP_000639414.1, 99% wekBGlucosyltransferase WcmS; UDP-Glc: Glc-Rha(Ac)-GlcNAc- UPPβ-1,6-glucosyltransferase (E. coli O158), ADN43874.1, 40% Wzy O antigenpolymerase Wzy (E. coli), ADR74237.1, 30% wekC Glycosyltransferase WfbF;UDP-Glc: FucNAc-GlcNAc-UPP α- 1,3-glucosyltransferase (E. coli),ABG81807.1, 46% fnlA UDP-N-acetylglucosamine- UDP-N-acetylglucosamine4,6- 4,6-dehydratase/5-epimerase dehydratase/5-epimerase (E. coli),WP_001556096.1, 95% fnlB UDP-2-acetamido-2,6- FnlB (E. coli),AAY28261.1, 97% dideoxy-beta-L-talose 4- dehydrogenase fnlCUDP-N-acetylglucosamine 2- UDP-N-acetylglucosamine 2-epimerase epimerase(E. coli) WP_000734424.1, 98% wbuB Glycosyltransferase UDP-L-FucNAc:GlcNAc-UPP α-1,3-N- Acetylfucosaminyltransferase (E. coli) P12b, O26],YP_006169152.1, 73% wbuC Truncated glycosyltransferase WbuC (E. coli),AAV74548.1, 72%

Comparisons of structure and gene cluster imply that all functionsneeded for assembly of the O25A OPS are encoded within the rfb clusterlocated between galE and gnd. The functions of the various enzymes ofthe rfb gene cluster (see FIG. 2A) are as follows:

RmlBDAC encode the enzymes required for biosynthesis of dTDP-L-Rhamnose,which is the substrate for the addition of L-Rha branch to the OPSrepeat unit.

FnlABC encode the enzymes required for biosynthesis of UDP-L-FucNAc,which is the donor substrate for the addition of L-FucNAc to the O25 OPSrepeat.

WekABC and wbuBC are glycosyltransferases according to homologyanalysis.

However, wbuC appears short and truncated and is unlikely functional.Thus, the most likely functional annotation indicates that there arefour glycosyltransferases generating the four linkages for assembly ofthe repeat unit.

Wzx and Wzy are required for flipping of the BRU to the periplasmicspace and their polymerization on Und-PP.

All functions required to synthetize the published O25A repeat unitstructure are encoded by the E. coli E47a and 83972 rjb clusters. Thusit was concluded that the rfb cluster is responsible for encoding theO25A OPS.

O25B

In 2009, clinical E. coli isolates from a Spanish hospital setting werecharacterized to determine clonal groups. See Blanco, et al. (2009) JAntimicrob Chemother 63, 1135-1141. Characterization of a) the ESBLtype, b) the O serotype, c) virulence genes, d) multi locus sequencetyping (MLST), and e) pulsed field gel electrophoresis typing (PFGE) wasdone. Results indicated that about 20% of all isolates could beattributed to the same clone: Serotype and MLST O25:H4 ST131, ESBL typeCTX-M15, Phylogroup B2, encoding a specific set of virulence genes. Theanalysis of the rfb cluster components of representative clinicalisolates showed an unknown 3′ sequence when compared to the typingstrain sequence from the E47a strain, and also from clinical isolatesidentified by an allele specific PCR typing method (See Clermont et al.,2008, J Antimicrob Chemother. 61(5):1024-8.; Clermont et al., Diagn.Microbiol Infect Dis. 2007, 57(2):129-36.; and Li, et al., 2010, JMicrobiol Methods 82, 71-77. In 2013, Phan et al. published the genomesequence of clone O25b:H4 STI31, confirming that the clone is a K-12derivative in agreement with the structure of its waa gene cluster asreported earlier. See Phan et al., 2013, PLOS Genetics 9(10):1-18(e1003834). Together, the data suggests that a novel O25 agglutinatingclone had emerged in E. coli isolated from hospital settings, and thatthe clone had specific ESBL, MLST, and PFGE phenotypes and contained analtered O antigen gene cluster.

PCR Typing

To determine whether the O25B serotype was present among the isolated E.coli strains identified in the epidemiology study described in Example1, O25 agglutination positive strains were analyzed by typing PCR forO25 and O25B. PCR was performed using colonies picked from a petri dishas template DNA source and different oligonucleotide primers.O25-specific primers, based on amplification of E47a O25 wzy, anddescribed in Li, et al. (2010) J Microbiol Methods 82, 71-77 were used.Also used were the O25B-specific primers described in Blanco, et al.(2009) J Antimicrob Chemother 63, 1135-1141, which are specific for anundefined 3′ portion of the O25b rfb cluster (LNB220). According to Phanet al., 2013, this O25B specific oligonucleotide pair anneal in a 3′portion of the O25B rfb cluster.

Of 24 tested clinical isolates with an O25 agglutination positivephenotype, 20 were assigned to the O25B serotype by PCR typing, whilethe remaining 4 were positively identified as belonging to the O25Aserotype by PCR typing. Thus, surprisingly, strains of the O25B serotypewere determined to be more frequent among the analyzed strains thanstrains of the O25A serotype.

Cluster Sequencing

To analyze the O25B rfb cluster genetically, the cluster of an O25BPCR-positive strain, designated “upec138” was sequenced. The genesidentified and their closest relevant protein homologs along withsuggested nomenclature are listed in Table 3, below. Genes specific forO25B and absent in O25A are indicated with an asterisk.

TABLE 3 O25B O antigen gene predictions from the rfb cluster. Mostmeaningful Gene homology/Protein[organism], name Putative Functionaccession, max. identity (BLAST) rmlB dTDP-Glucose 4,6- rffG geneproduct [E. coli NA114], dehydratase YP_006139244, 99% rmlDdTDP-6-Deoxy-D-glucose dTDP-4-dehydrorhamnose reductase 3,5-epimerase[E. coli NA114], YP_006139243, 100% rmlA Glucose-1-phosphate rffH2 geneproduct [E. coli NA114], thymidylyltransferase YP_006139242, 100% rmlCdTDP-4-dehydrorhamnose dTDP-4-dehydrorhamnose 3,5- 3,5-epimeraseepimerase [E. coli NA114], YP_006139241, 99% Wzx Wzx, O antigen flippaseWzx [E. coli strain E47a], ADI43260, 99% wekA Glycosyltransferase (GT)dTDP-Rha: Glc-Rha(Ac)-GlcNAc-UPP α-1,3-rhamnosyltransferase [E. coli83972], ZP_04004894, 93% wekB Glucosyltransferase (GT) UDP-Glc:Glc-Rha(Ac)-GlcNAc-UPP β- 1,6-glucosyltransferase [E. coli 83972],YP_006106413, 93% Wzy O antigen polymerase membrane protein [E. coli83972], YP_006106412, 94% wbbJ* O-acetyl transferase O-acetyltransferase [E. coli 83972], YP_006106411, 95% wbbK* Glycosyltransferase(GT) UDP-Glc: Rha-GlcNAc-UPP α-1,3- glucosyltransferase [E. coli K-12],AAB88407, 60% wbbL* Glucosyltransferase (GT) lipopolysaccharidebiosynthesis protein, C-ter fragment, truncated protein [E. coli DH1],YP_006129367, 62%; dTDP- Rha: GlcNAc-UPP α-1,3- rhamnosyltransferase

The cluster composition rfb shows clear differences to the compositionof the O25A cluster. The genes in the 5′ portion of the cluster areclose homologs of each other (rmlD to wzy; E. coli E47a and 83972). Thisis not surprising for the rml genes which are homologous in many E. colistrains that synthetize L-rhamnose. Homology of the gene products ofO25A and B reaches into the wekC (025A) gene, before it drops to levelsbelow 25% identity, indicating unrelatedness of the protein sequences.See FIG. 3B. Further, it was found that the UDP-N-acetylfucosaminebiosynthesis genes of O25A are absent in strain upec138 (O25B), as aretwo glycosyltransferases downstream of fnlABC. See FIG. 3B. Takentogether this data suggests that O25B strains are unable to synthetizeUDP-L-FucNAc, except that L-FucNAc biosynthesis genes could be encodedoutside the rfb cluster. However, there is not a single case reportedfor an fnlABC locus outside the rfb cluster when L-FucNAc is present inthe BRU of the O antigen. Thus it is unlikely that strain 138 is able tosynthetize the published O25A basic repeat unit (BRU).

The genes identified in the O25B rfb cluster that are not present in therfb cluster of serotype O25A encode two glycosyltransferases and anO-acetyltransferase. These three genes share the same organization andthe encoded proteins have high homology with the wbbJKL genes found andcharacterized in K-12 strains of E. coli of the O16 rjb clustergenotype. See FIG. 3B. According to the genetic relatedness between theO25B and O16 serotypes, the nomenclature of the O16 rfb genes, wbbJKL,was applied to the homologous genes identified in the O25B rfb cluster.

The structure of the O16 BRU is known and the gene functions of wbbJKLhave been determined. See Steveneson et al., (1994) J Bacteriol.176(13):4144-56. WbbJKL are responsible for acetylation of L-Rhamnose,transfer of a D-Glc residue to L-Rha-D-Glc-UndPP, and the transfer ofthe L-Rha to D-GlcNAc-UPP, which is formed by wecA from the ECA cluster.Based on homology with O16 WbbJKL, and the known functions of O16WbbJKL, it was deduced that the O25B rfb cluster synthesizes a structurecontaining partially O16 and partially O25A elements together. It wasreasoned to be highly likely that WbbJKL_(O25B) synthesize the samestructure as WbbJKL_(O16), i.e. α-D-Glc-1,3-α-L-Rha(2Ac)-1,3-a-D-GlcNAc.This trisaccharide structure is identical to the unbranched ‘core’backbone of O25A with the only exception that the L-Rha(2Ac) replacesthe L-FucNAc. The replacement would accordingly be a conservative one,as D-FucNAc and L-RhaOAc are both monosaccharides with a 6-deoxy and2-acetyl function. The only difference is conformation, as fucose isrelated to galactose, and rhamnose to mannose, resulting in a differentorientation of the OH group at position 3 and the methyl group atposition 5. Linkages between the monosaccharides would be identical (alla-1,3), indicating that the structures would be similar in shape andchemical characteristics. In analogy, the proteins encoded in theupstream part of the O25A and B rfb clusters (rmlDCAB and wekAB) branchthe BRU of O25A or B by attachment of the branching D-Glc and L-Rha toeither ‘core’ backbone trisaccharide. This would mean they accept eitherbackbone (with L-FucNAc OR L-Rha(2Ac)) as a substrate.

The presence of L-Rha as the second monosaccharide from the reducing endof the O25B BRU explains why the L-FucNAc biosynthesis can be absent inO25B. UDP-L-FucNAc is not needed, because it is replaced by thedTDP-L-Rha biosynthesis genes that are present in the 5′ end of thecluster (rmlDBAC).

Phan et al., 2013 did a similar genetic analysis on an O25B clinicalisolate, but concluded differently. They also sequenced the entiregenome to search for the UDP-FucNAc biosynthesis gene cluster; however,they state that the machinery for UDP-L-FucNAc in strain O25B:H4 STI31EC958 is missing not only in the O25B rfb cluster, but in the entirestrain. However, Phan concluded that UDP-L-FucNAc must be synthetized ina different way, assuming that O25B:H4 STI31 EC958 makes the same Oantigen structure as E47a, i.e., O25A. Instead, it is disclosed hereinthat the most likely scenario is that O25B strains cannot make L-FucNAc,but instead replace the second residue of the BRU with an O-acetylatedL-rhamnose residue, and the genes required for this change areexclusively encoded in the rfb cluster.

In addition, the presence of an O-acetyl transferase homolog in the O25Bcluster suggests O-acetylation in the O25B BRU, a modification absent inO25A. Accordingly, it was determined that the structures of O25 antigenfrom serotypes O25A and O25B must be different.

O25B Structural Analysis

To confirm the hypothesis of a different O25 antigen structure, thechemical composition and arrangement of the O antigens of the O25clinical isolates described in Example 1 were analyzed. To characterizethe O25 OPS structures in more detail, several methods were used.

First, the O antigen structure was analyzed by SDS PAGE.Lipopolysaccharide (LPS) from clinical isolates was analyzed fordifferences in electrophoretic mobility using different staining methodsafter SDS PAGE. To visualize LPS amounts, silver staining and anti-O25specific Western blots were performed. See FIGS. 5A-5B, which depictsresults of the analysis of 10 isolates. The data shows that similarsignal intensities are obtained by silver staining of the different LPSpreparations. In contrast, probing with the specific antiserum showedstronger signal intensities in 3 out of 10 samples (isolates upec436,upec767, upec827). It was speculated that the different signalintensities arose due to differences in structure of the OPS.

To elucidate the O25B structure in detail, different analytical methodswere applied. Clinical isolate upec138 was positive for the O25B by PCR,and exhibits a weaker recognition by the O25 agglutination antiserumthan O25A strains. See FIGS. 5A-5B. In addition, the strain is ESBL, butsensitive to FOS, IPM, and TZP, and resistant to AM, CXM, NOR, and CIP.Another clinical isolate, strain upec436, was negative for O25B by PCR,but positive for the general O25 (O25A) by PCR. upec436 also was foundto be strongly reactive with the O25 agglutination antiserum when itsLPS was analyzed by Western blotting. See FIGS. 5A-5B. 5. LLO from bothstrains was extracted, labeled with a 2AB and analyzed by normal phaseHPLC. See FIG. 6; LLO of upec138 and upec436, 9.079 and 9.081). Theelution patterns showed clear differences between the two extracts.MS/MS analysis of strain specific peaks detected signals compatible withthe expected BRU structures.

Signals in strain upec436 (9.081): The peak at 62′ elution time wasanalyzed by MS and found to contain as the main mass a molecule withm/z=1021 Da, i.e. a molecule corresponding to the expected mass of thecomplete O25A OPS BRU. MS/MS produced a fragmentation pattern compatiblewith the monosaccharide sequence of O25A (FIG. 7A; MS/MS of m/z=1021).

Signals in strain upec138: The main mass in the peak at 50′ elution timewas m/z 1022 Da, i.e. one Da more than the complete O25A repeat unit.MS/MS analysis (FIG. 7B; O25B MS/MS) showed fragmentation behavioralmost identical to the O25A repeat unit, and localized a 1 Dadifference to the 2^(nd) monosaccharide from the reducing end(identified by a fragmentation Y ion of m/z=551 in O25A MS/MS, andm/z=552 in strain upec138). An additional peak eluting at 60′ showedsimilar fragmentation, but a 42 Da difference in the mother ion mass(m/z=980) that localized to the same monosaccharide (m/z=510), i.e. thesecond one from the reducing end. Interpretation of these results isgiven below.

The OPS extraction, hydrolysis and 2AB labeling procedure involves acidtreatment to remove the Und-PP from the OPS. It was shown that thetreatment conditions partially remove O-acetylation, but notN-acetylation. Thus, it is likely that the peak at 60′ represents adeacetylated BRU mass that emerged from the chemical hydrolysis of thematerial in the 50′ peak. Taken together, this data indicates that thereis an O-acetylation in O25B at the same monosaccharide position as thereis N-acetylation in the L-FucNAc of O25A.

To confirm chemically that the acetylation at the second residue fromthe reducing end is O-linked, a deacetylation assay was performed. TheO25B specific peak from 2AB LLO HPLC at 50′ elution time was collectedfrom an O25B PCR positive strain and treated with alkali described under‘Methods’. Re-analysis by HPLC resulted in a peak at 60′ elution time asidentified in a O25B peak from FIG. 6, containing a main mass ofm/z=979, with a MS/MS fragmentation ion pattern consistent with an O25BBRU that had lost its O-acetyl group. N-acetyl groups are stable towardsalkali treatment as shown by the remaining N-acetyl group in thereducing end D-GlcNAc in the same molecule.

In conclusion, it was determined that the O25B representative strainupec138 is structurally and genetically related to the O25A and O16 OPS(FIGS. 3A and B) from E. coli. O25B differs from O25A in having a repeatunit structure containing an O-acetyl group instead of an N-acetyl groupat the second monosaccharide of the repeat unit, which is a L-Rharesidue and not a D-FucNAc. These changes were most likely caused by areplacement of the UDP-FucNAc biosynthesis machinery and the D-FucNActransferase by a DNA stretch encoding two glycosyltransferases and anO-acetyltransferase. These genes are related to the O16 gene cluster,based on homology and functionality analysis. The final structures aredifferent, but similar, explaining the cross-reactivity observed withthe O25 agglutination antiserum.

As discussed above, it was concluded and proposed based on analysis oftheir rib clusters that O25A OPS contains L-FucNAc, whereas thestructure is absent in O25B. To investigate if FucNAc is absent fromO25B, monosaccharide composition analysis of EPA bioconjugates producedin O25A and O25B strains was performed (FIG. 9) using the PMP labelingmethod and HPLC analysis method described above. To producebioconjugates, clinical E. coli isolates with the O25A and O25Bphenotypes were prepared, and modified for optimal bioconjugateproduction. As part of the modification, the waaL genes from strainsupec_436 (O25A) and uepc_138 (O25B) were deleted as previously described(see Datsenko and Wanner, (2000) Proc Natl Acad Sci USA 97, 6640-6645)informed by the method for core type determination (see Amor, et al.,(2000) Infect Immun 68, 1116-1124). Resulting strains were transformedwith expression plasmids for 4S-EPA (pGVXN659) and an oligosaccharyltransferase, PglB (pGVXN939), for O25A production; and with pGVXN114 andpGVXN539 (producing 2S-EPA) for production of O25B bioconjugateproduction. O25B bioconjugates were produced in a 2 L shake flask withsubsequent affinity purification from periplasmic extracts by IMAC. O25Aconjugates were produced by fed-batch fermentation, and purified by atwo-step purification procedure starting from clarified whole cellhomogenate generated by high pressure homogenization as described in themethods section above. Monosaccharide composition analysis was performedas described above.

The results confirmed the absence of a signal for FucNAc in theO25B-derived bioconjugates, whereas the O25A-containing bioconjugatesshowed a peak at the expected elution time as determined by subjecting amix of monosaccharides to the same sample processing procedure as acontrol. It was thus confirmed that the putative structure of O25B is,as expected based on analysis of the rfb cluster, L-FucNAc-less.

The complete structure of the repeat unit (RU) of the O-antigenpolysaccharide (O-PS) from the Escherichia coli O25B O-antigen wasdetermined by nuclear magnetic resonance of the bioconjugate afterpartial enzymatic digestion of the EPA carrier protein moiety. Theanalysis confirmed that the O25B O-PS is composed of a pentasaccharideRU. The ¹H and ¹³C signals were assigned by 2D NMR correlationtechniques, which confirmed that the structure of the O25B O-PS RUdiffers from the published O25A O-PS RU structure (Kenne, L., et al.1983. Carbohydr. Res. 122:249-256; Fundin, J., et al. 2003. Magn. Reson.Chem. 41:202-205) by the substitution of an α-3-FucpNAc residue by anα-3-Rhap residue, with more than 90% of this residue being O-acetylatedat the C2 position. The complete O25B O-PS RU is shown below (O25B′):

Bioconjugate Production and Characterization

To analyze the O25A and O25B polysaccharide antigens further, morebioconjugate material was produced. For O25A, the purified batch ofO25A-EPA from above was used for further characterization experiments.For O25B-EPA production, a strain with a genomically integrated O25Bcluster was constructed: W3110 ΔwaaL ΔgtrABS ΔrfbO16::rfb(upec138), withplasmids pGVXN1076 and pGVXN970. This strain was constructed startingfrom W3110 by the methods of Datsenko and Wanner and a homologousrecombination technique for site directed integration of large insertsinto bacterial chromosomes (see International Patent Application No.PCT/EP2013/071328).

The resulting O25B bioconjugates were characterized using standardrelease and characterization assays. Bioconjugates were purified usingtwo consecutive anionic exchange and size exclusion chromatographysteps, yielding 97.2 and 98.1% pure O25A and O25B bioconjugatepreparations, respectively. SDS PAGE quantification was used for purityanalysis. See FIG. 10 (O25A) and FIG. 11 (O25B). Sugar to protein ratioswere calculated based on sugar quantification by the anthrone assay (seeLaurentin and Edwards, (2003) Anal Biochem 315, 143-145) and the BCAassay for protein concentration, resulting in 40.2 and 26.6% for O25Aand O25B bioconjugates. Analytical size exclusion chromatography showeda monomeric state of the particles in agreement with the expectedhydrodynamic radius of EPA with attached glycan chains.

Applications

To address the immunogenic potential of the O25B structure, severalpreclinical experiments using O25B and O25A bioconjugates wereperformed. As was shown in FIGS. 5A and 5B, all clinical isolatesidentified as O25 positive in Example 1 (i.e., both O25A and O25Bisolates) were positive with O25A antisera commonly used to detect O25serotypes (typing sera from the O25A strain E47a) in Western blots.Thus, anti O25A antiserum appears to be cross reactive to the LPS fromO25B strains. To analyze the antibody response and cross reactivity indetail, O25 bioconjugates were produced. Maltose binding protein (MBP)was used as a carrier protein, and the carrier protein was linked toO25A or O25B. Table 4 depicts the strains used for protein production.The used strains were identified by PCR for their O25A or B genotype.Expression was performed in TB medium and protein product purified fromperiplasmic extracts.

TABLE 4 pglB Carrier Purification Bioconjugate Strain plasmid plasmidprocedure MBP-O25A upec436 pGVXN939 pGVXN659 Q, A ΔwaaL::kanR MBP-O25Bupec350 pGVXN939 pGVXN659 Q, A, S ΔwaaL::clmR Q: Resource Qpurification; A: Amylose resin; S: Size exclusion chromatography

Immunizations using the obtained bioconjugates were performed using astandard rabbit immunization protocols (the eurogentech 28-day speedyprotocol). 50 μg of polysaccharide bound to MBP, were injected at days0, 7, 8, and 18 with a proprietary Freund's free immunostimulatorycompound. The resulting final bleed antisera obtained at day 28 afterthe first immunization were tested for their specificity towards O25A orO25B LPS. FIG. 22 shows a comparison of the antisera reactivitiestowards the respective LPS (025A or O25B). LPS was prepared from upec436and upec138 by proteinase K digestion of whole cell samples in SDS-PAGELämmli buffer. The same amount of LPS was loaded in two SDS-PAGE gelsfollowed by electrotransfer to nitrocellulose membranes and detectionusing O25A and O25B antisera. The results show that the O25A antiserumrecognizes the O25A LPS better than O25B LPS, while the O25B antiserumrecognizes the O25B LPS better than O25A LPS. This result indicates thatthe autologous antigen makes a better antigen. Thus, inclusion of O25Bantigen into a vaccine will provide better protection against thepredominant O25B clinical strains of the O25 group than the O25Aantigen.

Example 3: E. coli O1

Structural databases list different subserotype structures for E. coliO1. In particular, O1A, O1A1, O1B, O1C. O1A and O1A1 are structurallyidentical and believed to be associated with disease, although O1B and Chave not been reported to be pathogenic (see Gupta, et al., (1992) JBacteriol 174, 7963-7970), and represent a minority among O1 isolates.Structures of O1A/O1A1, O1B, and O1C are shown in FIG. 12B. To analyzethe O1 subserotype distribution in the UPEC epidemiology study ofExample 1, the O antigen structures of several clinical isolates fromthe study were analyzed in detail. First, LPS structure of 12 strainsdetermined to be positive for O1 by agglutination assay were analyzed bySDS PAGE. See FIGS. 13A and 13B: O1 silver staining and Western blot.

Silver staining showed typical LPS signals in all lanes containingextracts from the O1 clinical isolates. Strong staining at anelectrophoretic mobility of about 10-15 kDa depicts the lipid A core,and ladder like signals with slower mobilities represent the lipid Acore modified with carbohydrate polymers composed of different numbersof O antigen repeat units. When the LPS from different isolates arecompared, differences appear in the modal length distribution, theelectrophoretic mobility of individual bands, and the ladder pattern.Based on these observations, three groups can be identified: (i) mostisolates (upec002, upec010, upec032, upec140, upec108, upec143, upec276,upec399, and upec425) exhibited indistinguishable electrophoreticmobility of individual bands, only differing in signal intensity andaverage chain length (modal length distribution); (ii) two isolates(upecl 19 and upec256) appeared to have slightly faster mobility inevery repeat unit LPS band, suggesting a different structure, e.g. adifferent modification of the lipid A core; and (iii) signals obtainedfrom isolate upec1096 appeared as a smear rather than a ladder,indicating a different OPS structure. See FIG. 13A.

Analysis by Western blotting and detection using the anti O1 antiserumshowed that LPS from all but upec1096 is detected by specific O1antibodies, indicative of cross reactive LPS molecules. This means that11 of the isolates are O1, and that upec1096 is most likely not an O1isolate (i.e., it was a false positive by agglutination assay).

To analyze the structural similarity of the O1 antigens in detail, 2ABlabeling of LLO and a high resolution normal phase HPLC technique wereused as described above. FIG. 14A shows an overlay of the chromatogramsobtained from 5 of the 11 clinical isolates. The fingerprinting area ofthe OPS appears at retention times of 110 to 150 minutes. The profilesindicate that all samples have signals appearing at the same retentiontimes, indicating identical molecule structures. Differences observedwere the intensity distribution, i.e. the elution time of the meanmaximum signal, and the general signal intensities. The remaining 6extracts resulted in peaks at the same elution times with differences inintensities. Only sample upec1096 was different with respect to the peakpattern, confirming the structural difference noted above.

MS/MS analysis of individual peak contents by MALDI-TOF/TOF analysis wasused to identify the sequence of monosaccharides in the O1 samples (seeFIG. 14B). MS analysis was performed from samples extracted not fromclinical isolates but from a W3110 ΔwaaL strain containing a cosmid withthe rfb cluster of upec032. Peaks eluting at 50, 80, 96, and 108 minuteselution time contained main masses of m/z=1021.4, 1849.6, 2693.9,3540.4. Fragmentation ion series obtained after MS/MS were consistentwith 1, 2, 3, and 4 repeat units of a HexNAc, three deoxyhexoses, and abranching HexNAc. This data can only be explained by the O1A subserotypestructure. The described peak series represents the O1 OPS attached toUPP in clinical isolates, and every consecutive peak differs to theprevious one by one repeat unit.

This data confirms the statements from the literature that therepresentative structure for the O1 O serotype of E. coli in theclinical UTI isolates from the study described in Example 1 is subtypeO1A.

To produce a bioconjugate carrying the O1A polysaccharide, W3110 E. colistrains were engineered to express the O1A OPS. Resulting strains wereW3110 ΔrfbO16::rfbO1 ΔwaaL, containing the rfb cluster of an O1 positiveclinical isolate (GU299791, cluster ranging from rmlB-wekO). The O1A OPSexpressing host strains were constructed by homologous recombination.The rfb cluster of an O1A clinical isolate was amplified using PCRoligonucleotides annealing in the DNA flanking the rfb cluster. Theamplified DNA was then used to replace the endogenous O antigen clusterof the well characterized laboratory strain W3110 by the homologousrecombination described in International Patent Application No.PCT/EP2013/071328. Expression plasmids for the carrier proteins pGVXN659and for PglB (pGVXN114, 939, 970, 971) were inserted by transformationand O1 expression was confirmed (see FIG. 15 and FIG. 16).

In a separate experiment, the clinical O1 isolate upec032 was engineeredto produce bioconjugates. Engineering required that antibiotic sensitivephenotype of the clinical isolate be considered. upec032 ΔwaaL wasconstructed and transformed with pGVXN939 and pGVXN579 for bioconjugateproduction using MBP as carrier protein. The advantage of using MBP andEPA as carrier proteins is the possibility to raise antisera with bothresulting in antisera that are crossreactive towards the polysaccharidecomponent but not the carrier. Such antisera are useful tools forevaluation of preclinical experiments, e.g. as coating agents to developpolysaccharide specific ELISA assays.

Example 4: E. coli O6

The E. coli O6 serotype is the most frequent ExPEC reported to date(George, D. B., and Manges, A. R. (2010) Epidemiol Infect 138,1679-1690). Not only the study described in Example 1, but also datataken from the literature confirms that the O6 serotype is among the topfour serotypes in many ExPEC caused manifestations (see FIG. 4).

Two structures of the O6 OPS have been reported in the literature (seeJann et al., Carbohydr. Res. 263 (1994) 217-225, and Jansson et al.,Carbohydr. Res. 131 (1984) 277-283). The structures of the reported O6antigens are shown in FIG. 17B. They are identical except for thebranching monosaccharide of each, which is either Glc or GlcNAc.However, the literature has not identified the predominant O6 structurein clinical isolates involved in UTI.

To choose the most representative structure of the O6 antigen forvaccine purposes, the OPS structures of O6 agglutination positiveclinical E. coli isolates from the study of Example 1 was investigatedusing the same approach as described above for the O1 serotypes. Silverstaining and Western blotting using anti O6 antiserum identified one of12 clinical isolates not reactive to anti O6 serum, although LPS wassilver stained in all samples (not shown), suggesting a false positiveagglutination result. However, it is likely that Glc or GlcNAcdifferences would not be detected by electrophoretic mobility shift ongels.

For detailed structure analysis, LLO fingerprinting was used. As areference for either of the two reported structures, extracts fromstrains with reported branching Glc (CCUG11309) and GlcNAc (CCUG11311)were included in the analysis. Comparison of the two HPLC traces showpeaks series eluting at 70.8, 103.3, and 122.2′ for CCUG11309 derivedsamples, and series of 68.8, 100.3, and 118.3 for CCUGI 1311 samples.See FIG. 18A. Peaks were analyzed by MS for the main masses present inthe peaks and MS/MS for the monosaccharide sequence of these mainmasses. The data confirmed for the CCUG11311 extract derived peak seriesm/z=1094.4, 2027.6, and 2962 (MSO154), corresponding to GlcNAc branched1, 2, and 3 BRU polymers as expected. m/z=1053.4, 1945.7, and 2836.9with branching Glc were identified previously in extracts from a W3110strain expressing the cloned rfb cluster of CFT O6 clinical isolate,having identical 2AB fingerprint peak elution times as CCUG11309(MSO138). When chromatograms obtained from the 12 clinical isolates werecompared to the reference strains, 11 signals contained the peak seriesindicative of the O6 OPS with a branching Glc residue. Five of these 11chromatograms are shown in FIG. 18B. The one sample not generatingsignals at O6 specific elution times was not O6, i.e. most likely afalse positive from the agglutination test. Thus, the O6 OPS with a Glcbranch (FIG. 17B, top) is the most representative structure among the O6serotypes isolated from the epidemiology described in Example 1.

To produce a bioconjugate carrying the O6Glc polysaccharide, W3110 E.coli strains were engineered to express the O6 OPS by replacing theW3110 rfb cluster with the rfb cluster from strain CCUG11309. See Tables7 and 13. Resulting strains were W3110 ΔrfbO16::rfbCCUG11309 ΔwaaL,containing the rfb cluster of an O6 positive E. coli strain withreported Glc branch in the BRU (see above). The O6Glc OPS expressinghost strain were constructed by homologous recombination. The rfbcluster was amplified using PCR oligonucleotides annealing in the DNAflanking the rfb cluster. The amplified DNA was then used to replace theendogenous O antigen cluster of the well characterized laboratory strainW3110 by the homologous recombination described in International PatentApplication No. PCT/EP2013/071328. Expression plasmids for the carrierproteins and for PglB were inserted by transformation and expression ofthe expected OPS on EPA was confirmed by Western blotting.

Example 5: E. coli O2

The repeat unit structure of the O2 polysaccharide has been known since1987 (Jansson, et al., (1987) Carbohydrate research 161, 273-279). It isshown in FIG. 19B. Two O2 O antigen gene cluster sequences are availablefrom public databases (GenBank EU549863 and GU299792). Comparativeanalysis has been made and glycosyltransferase activities have beensuggested (Table 5; Fratamico et al., 2010, Canadian journal ofmicrobiology 56, 308-316; and Li, et al., (2010) J Microbiol Methods 82,71-77).

TABLE 5 O2 O antigen cluster gene predictions from the rfb cluster aspublished by Li, et al. and Fratamico, et al. is indicated in brackets.Most meaningful Gene homology/Protein[organism], name Putative Functionaccession, max. identity (BLAST) rmlB dTDP-Glucose 4,6- dTDP-Glucose4,6-dehydratase dehydratase (E. coli) IAI39), YP_002406996.1, 98% rmlDdTDP-6-Deoxy-D-glucose dTDP-6-Deoxy-L-mannosedehydrogenase 3,5-epimerase(E. coli, ACA24825.1, 97% rmlA Glucose-1-phosphate Glucose-1-phosphatethymidylyltransferase thymidylyltransferase (E. coli IAI39),YP_002406998.1, 99% fdtA NDP-hexose isomerase NDP-hexose isomerase(Yersinia intermedia ATCC 29909), ZP_04635116.1, 67% fdtC WxcM-likeprotein Hypothetical protein PROVRETT_01740 (Providencia rettgeri DSM1131), ZP_03638653.1, 71% fdtB Aminotransferase WblQ protein(Photorhabdus luminescens subsp. laumondii TTO1), NP_931971.1, 65% Wzx Oantigen flippase Polysaccharide biosynthesis protein (Pectobacteriumcarotovorum subsp. carotovorum PC1), YP_003016888.1, 50% wekPGlycosyltransferase (GT) Hypothetical protein FIC_01940 (wegQ)(Flavobacteriaceae bacterium 3519-10), YP_003096444.1, 29% rmlCdTDP-4-dehydrorhamnose RmlC (E. coli), ACA24796.1, 70% 3,5-epimerase WzyO antigen polymerase Hypothetical protein Gura_3055 (Geobacteruraniireducens Rf4), YP_001231799.1, 26% wekQ GlycosyltransferaseGlycosyl transferase, putative, gt2D (wegR) (Cellvibrio japonicusUeda107), ref|YP_001983904.1, 31% wekR Glycosyltransferase Glycosyltransferase, group 1 (Shewanella frigidimarina NCIMB 400), YP_751504.1,57% wekS Sulfatase Putative transmembrane sulfatase protein (wegW)(Stenotrophomonas maltophilia K279a), YP_001970541.1, 39%

Comparison of structure and gene homologies indicated that all functionsfor biosynthesis of the polymer are present:

rmlBDAC encode the enzymes required for biosynthesis of dTDP-L-Rhamnose,which is the substrate for the addition of L-Rha to the backbone byglycosyltransferases wekPOR;

fdtABC provide dTDP-D-Fuc3NAc for the branching glycosyltransferases;

wzy and wzr homologs responsible for flipping of the Und-PP-bound repeatunit from the cytoplasm to the periplasm; and

wekPOR, are predicted glycosyltransferases, and are predicted to formglycosidic linkages of the O2 BRU (three L-Rha and one L-FucNAc).

The wekS gene found in the published O2 rfb cluster sequences is apredicted membrane bound sulfatase, and thus most likely is not involvedin BRU formation. This would mean that—if one assumes the one enzyme onelinkage rule—that one enzyme among the group of wekPOR must bebifunctional to provide the four glycosidic linkages.

That the one enzyme—one linkage rule is not absolute was shown inmultiple examples in which less glycosyltransferases than linkages areknown, e.g., in Shigella flexneri Y, S. flexneri 6, C. jejuni, and E.coli O1A. In these examples, multifunctional glycosyltransferases areresponsible for the formation of more than one glycosidic linkage, theyare ‘bi-’ or ‘multi-functional’. Always, it is the same monosaccharidewhich is added multiple times. Repeated rhamnose residues—as found inserotype O2—are often associated to such multi-functional enzymes.

Due to the presence of truncated transposon elements flanking the wekSsequence, it has been speculated that the wekS locus was inserted intothe rfb cluster by a DNA recombination event (see Fratamico et al.,2010, Canadian journal of microbiology 56, 308-316). Atransposon-mediated insertion of the wekS locus would suggest that theO2 OPS biosynthesis did exist without wekS presence before, andaccordingly wekS would not be required for the synthesis of the O2 OPSpolymer. To confirm this hypothesis, O2 OPS formation was reconstitutedin a recombinant expression system a ‘clean’ genomic background,containing an O2 rfb cluster lacking the wekS gene. To achieve this, theO antigen cluster from strain W3110 was replaced by the rfb cluster fromthe O2 positive strain upec116 lacking the wekS DNA. Chromosomalreplacement was done by homologous recombination as described inInternational Patent Application No. PCT/EP2013/071328. The resultingstrain was W3110 ΔwaaL ΔrfbW3110::rbO2 ΔwekS. OPS was prepared andanalyzed by 2AB labeling and normal phase HPLC and fluorescencedetection as done for O1A and O6 OPS (see above) and analyzed by normalphase HPLC (FIG. 21) and compared to the signals from wild type strainCCUG25. Analysis resulted in a series of overlapping peaks between 40and 140 minutes elution time.

MS analysis of the rfbO2 cluster dependent peak series showed mainmasses with the same differences among consecutive peaks (i.e. of asingle O2 RU).

MS analysis of the molecules collected in the respective peaks wasperformed to analyze the structure of the OPS. Peaks obtained at 43.5,73.1, 81.4 and 90′ obtained from wild type strain CCUG25 were collectedand analyzed by MS and MS/MS. Masses and Y fragment ion seriescompatible with the expected 1, 2 and 3 repeat unit O2 OPS moleculeswere found (m/z=989.4 (FIG. 23), 1817.8, 2646.1, all Na⁺ adducts).

To confirm the O2 OPS from clinical isolates, 12 clones were analyzedfor their OPS structure as described above for the O1 serotype. First,LPS was prepared to be analyzed by silver staining and Western blotting.The results are depicted in FIGS. 20A and 20B. All samples showed aladder-like banding pattern with two apparent different mean ladderlengths. Anti-O2 antiserum detected all LPS samples, indicating thatagglutination correctly identified all isolates as O2 serotypes.

To produce a bioconjugate carrying the O2 polysaccharide, W3110 ΔwaaLΔrfbW3110::rfbO2 ΔwekS was used. Expression plasmids for the carrierproteins and for PglB were inserted by transformation and expression ofthe expected OPS on EPA was confirmed by Western blotting. See Tables 7and 13 and above.

Example 6: Immunological Analysis of the Different O Antigens

To assess the immunological potential of bioconjugates containing theselected antigenic polysaccharides, a preclinical study was performed.O1A-EPA, O2-EPA, O6Glc-EPA and O25B-EPA bioconjugates were produced,purified, and characterized as described above and in the methodssection.

TABLE 6 Overview of the preclinical rat study, including vaccine groupnumbers, group sizes, vaccines used, and quality indication of thevaccine preparation, Production Injection Number of Treatment Purity/strain/(Strain/ Group route animals [0.2 μg PS] % S/P ratio/%plasmid/Plasmid) 1 i.m. 8 O1-EPA^(tetra-pool) 97 22 upec032 ΔwaaL::kanR/[0.2 μg PS] pGVXN939/ pGVXN659 W3110 Δrfb::rfb(upec116)(ΔwekS) 4 i.m. 8O2-EPA^(tetra-pool) 90 34 ΔwaaL::clmR/ [0.2 μg PS] pGVXN939/ pGVXN659 7i.m. 8 O6-EPA^(telra-pool) 84 38 W3110 ΔwzzE-wecG [0.2 μg PS] ΔwaaLΔwbbIJK ΔgtrS ΔwzxO16/ pGVXN348/ pGVXN114/ pGVXN659 9 i.m. 8O25B-EPA^(tetra-pool) 85 21 upec163 ΔwaaL [0.2 μg PS] pGVXN112/ pGVXN65910 i.m. 8 EPA^(tetra) W3110 ΔwaaL/pGVXN659 11 i.m. 8 TBS 12 i.m. O1 +O2 + O25B +  89*  29* Blend from above O6-EPA^(tetra) batches [0.2 μgPS/each] *calculated values

Purified bioconjugates were used to immunize 9 week old female SpragueDawley rats. 100 μl solutions of the same dose were injected intramuscularly (i.m.) at days 1, 22, and 43 into the rats which wereterminally bled and sacrificed at day 56.

Different groups of rats received different vaccines: alwaysunformulated, comprising bioconjugates alone or in combination asindicated in Table 6. ELISA plates coated with the cognate LPS producedin a waaL positive strain were used to measure immunogenicity in theform of ELISA titer of the rat sera at the terminal bleeding timepointof 56 days after the first injection (FIGS. 25-28). Taken together,statistical significant immunogenicity was observed for all vaccinationgroups measured against controls (unglycosylated EPA or TBS buffer).Thus, the bioconjugates selected and produced represent useful vaccinecandidate compounds.

For all O-antigen-EPA conjugates tested, statistically significantimmunogenicity was observed for all vaccination groups measured againstcontrols (unglycosylated EPA or TBS buffer). Thus, the bioconjugatesselected and produced represent useful vaccine candidate compounds forthe induction of O-antigen-specific antibodies.

Example 7: Physico-Chemical Characterization of the Bioconjugates

The four bioconjugates described in the examples above (O-antigen ofO25B, O1A, O2 and O6, respectively conjugated to EPA as a carrierprotein) were prepared as monovalent batches (active pharmaceuticalingredients, APIs) or combined in a single preparation as a multivalentvaccine against ExPEC. Various batches were produced: pre-clinicalbatches, toxicity study batches, and clinical batches. Table 7 indicateshost strains used for the production of conjugates.

TABLE 7 Host strains for production of preclinical, toxicology study andclinical batches EPA PglB expression expression Product Strain plasmidplasmid EPA-O1A W3110 Δrfb::rfb(upec032) pGVXN1076 pGVXN970 ΔwaaL EPA-O2W3110 Δrfb::rfb(upec116) pGVXN1076 pGVXN971 ΔwaaL EPA- W3110Δrfb::rfb(CCUG11309) pGVXN659 pGVXN114 O6Glc ΔwaaL EPA- W3110Δrfb::rfb(upec138) pGVXN1076 pGVXN970 O25B ΔwaaL ΔgtrABS

The four monovalent pre-GMP batches and an un-glycosylated EPA referencestandard were thus analyzed by size-exclusion chromatography withmulti-angle light scattering (SEC-MALS), in order to quantify the degreeof mono- and di-glycosylation of the individual bioconjugates, and todetermine the molecular mass (MW) of the protein carrier and of the O-PSattached to it. The samples were separated on a TSKgel-G3000 SWxl columnin phosphate buffer (pH 7.0; 50 mM NaCl, 150 mM sodium phosphate) andmonitored by UV (214 and 280 nm), refractive index (RI) and multi anglelight scattering (MALS).

For the un-glycosylated EPA carrier protein, a MW of 63-67 kDa wasdetermined (theoretical MW of 70.5 kDa, based on amino acids sequence).In the bioconjugates, only EPA was detected at 280 nm, allowing its MWto be extracted from the total MW measured by RI and MALS: in thebioconjugates, the measured MW of the EPA moiety was 65-71 kDa.

The analysis of the pre-GMP API product standards is displayed in Table8, indicating the presence of mono- and di-glycosylated conjugates, witha MW in the range 75-79 kDa and 87-91 kDa, respectively. The O-PS partsfeatured a MW of 16-24 kDa for the di-glycosylated species, and 8-14 kDafor the mono-glycosylated species, respectively. Considering the MW ofthe RU of each serotype, an average number of 10-16 RU perpolysaccharide chain was determined, in good agreement withhydrazinolysis and MS data.

TABLE 8 SEC-MALS analysis of the monovalent pre-GMP API productstandards Peak 1 Peak 2 Total O-PS Total O-PS Monovalent batch MW MW MWMW EPA-O1A 15% 87 kDa 16 kDa 83% 75 kDa  8 kDa EPA-O2 29% 88 kDa 19 kDa70% 76 kDa 10 kDa EPA-O6 47% 88 kDa 21 kDa 50% 78 kDa 12 kDa EPA-O25B56% 91 kDa 24 kDa 42% 79 kDa 14 kDa Peak 1: di-glycosylated form. Peak2: mono-glycosylated form.

Circular dichroism (CD) analysis of an O25B bioconjugate batchformulated in Tris buffered saline (TBS), showed that formulations at pH6.8 to 7.4 had spectra similar to that of the un-glycosylated EPAcarrier protein, with a mixture of alpha helical and beta sheetstructures, as expected based on the published crystal structure of EPA.Therefore, based on these CD analyses, the glycosylation with O25B O-PSchains did not seem to affect the secondary structure of the EPA carrierprotein.

Differential scanning calorimetry (DSC) analysis of an O25B bioconjugatebatch formulation in TBS at pH 6.8 to 7.4, and in phosphate bufferedsaline (PBS) at pH 7.1 to 7.8, showed melting curves comparable to thatof un-glycosylated EPA, with a melting point of approximately 52° C.This result indicated that the biophysical characteristic of the EPAcarrier protein did not change upon modification with O25B O-PS chains.

Example 8: Stability of Monovalent Bulks and Tetravalent VaccinePreparations

Prior to large scale production, consistency of the manufacturingprocess was assessed on a small scale. Consistency batches wereevaluated in extensive stability studies that included accelerated andstress storage conditions to identify degradation pathways. Stability ofthe four monovalent vaccine components (APIs) was tested over a 3 monthperiod.

Analysis of stability data of pre-clinical APIs indicated stability overat least 3 months when stored at −75±15° C. (normal storage conditions).No statistically significant trends were observed at the intendedstorage condition by statistical linear regression analysis. Also at theaccelerated (+5±3° C.) and stress storage condition (+25±5° C.) theproduct was stable over at least 3 months, as evidenced by the lowvariability of stability-indicating parameters. The data for the O1Apre-clinical API is shown in Table 9. The three other serotypes (O2, O6,O25B) demonstrated similar stability data.

TABLE 9 Stability data of O1A pre-clinical API batch S/P S/P PurityPurity t0 3 mo. t0 3 mo. −75 ± 15° C. 19.3 19.8 98.1% 97.6%  +5 ± 3° C.20.9 98.6% +25 ± 5° C. 21.3 98.3% S/P: sugar to protein ration asdetermined by anthrone and BCA assays, respectively. Purity: asdetermined by reverse phase high resolution liquid chromatography(RP-HPLC).

Stability of the tetravalent vaccine composition (O25B, O1A, O2 and O6bioconjugates) was tested during over a 3 month period. The studiesincluded accelerated and stress storage conditions to identifydegradation pathways. The resultant data are considered to be relevantfor the initial justification of the GMP IMP (investigational medicinalproduct, the tetravalent ExPEC vaccine composition) shelf life.

Analysis of stability data of the tetravalent ExPEC vaccine pre-clinicalbatch indicated stability over at least 3 months when stored at +5±3° C.(normal storage conditions), as shown in Table 10. No statisticallysignificant trends were observed at the intended storage condition bystatistical linear regression analysis. Also at the accelerated storagecondition (+25±5° C.) the product was stable, as evidenced by the lowvariability of stability-indicating parameters.

TABLE 10 Stability data of pre-clinical tetravalent vaccine batch MW MWPurity Purity t0 3 mo. t0 3 mo.  5 ± 3° C. 302 & 192 294 & 188 98.3%98.7% kDa kDa 25 ± 5° C. 297 & 191 98.2% kDa MW (molecular sizedistribution): two main product peaks (i.e. mono- and di-glycosylatedspecies), as determined by size exclusion high resolution liquidchromatography (SE-HPLC). Purity: as determined by reverse phase highresolution liquid chromatography (RP-HPLC).

Together, these studies demonstrate that the APIs and the tetravalentExPEC vaccine composition were stable for at least three months, andthus are suitable vaccine compositions with respect to stability.

Example 9: Toxicity Study on Tetravalent Vaccine Preparation

Toxicity and local tolerance of a tetravalent vaccine preparation (O25B,O1A, O2 and O6 bioconjugates) following two intramuscularadministrations (quadriceps femoris was used for treatment) in SpragueDawley rats on days 1 and 14 was assessed. Reversibility, persistence,or delayed occurrence of any changes was assessed after a 14-dayrecovery period on day 28. Necropsy of the animals in the main groups(10 male and 10 female for both the vaccinated and the control group)occurred on day 17, and for the recovery groups (5 male and 5 female forboth the vaccinated and the control group) on day 28 (after a 14-dayrecovery period). This was not associated with any effects regarded asadverse that could be ascribed to treatment. The dose administered, i.e.a full human dose equivalent of 4 μg per O-antigen (16 μg totalO-antigen for the tetravalent vaccine), as administered on days 1 and14, was considered to be the no-observed-adverse-effect-level (NOAEL)for the tetravalent ExPEC vaccine under the conditions of this study. Inaddition, immunogenicity of the tetravalent ExPEC vaccine was confirmedat both day 17 and day 28, following assessment of the serum samples.Higher titers of anti-O1A, anti-O2, anti-O6 and anti-O25B IgG antibodieswere induced in the vaccinated group, compared to controls that receivedonly formulation buffer (25 mM Tris, 130 mM NaCl, 2.7 mM KCl, pH 7.4).

These data confirm that the tetravalent ExPEC vaccine has a suitabletoxicity profile for administration as a vaccine, and induces antibodiesto at least all four E. coli serotypes from the O-antigens present inthe vaccine (i.e., O25B, O1A, O2 and O6).

Example 10: Epidemiology of O-Serotypes Associated with Bacteremia

To determine O-serotype distribution among extraintestinal E. colicausing bacteremia in the elderly, an epidemiological study wasconducted on a panel of E. coli blood isolates collected from patientsolder than 60 years of age. In total, 860 blood isolates from the period2011-2013 were collected from subjects in the US, UK, Germany, Spain,and the Netherlands, and analyzed by classical O-agglutination. As shownin table 11, the O-serotype distribution of bacteremia isolatesresembled the O-serotype distribution found in patients suffering fromurinary tract infection (UTI, see Table 1A). Serotype O25 was mostprevalent in the bacteremia population studied; subtyping of fifty-sevenisolates by PCR showed that fifty-six (98%) of the O25 serotypes weretypeable as O25B. In both target populations (UTI and bacteremia),serotypes O1, O2, O6, and O25 were identified as the four most prevalentserotypes. Overall, these data confirm that serotype distribution amongboth urinary tract infection and bacteremia isolates is highly similarand independent of geographical location, time of isolation, andindication.

TABLE 11 Distribution of the most common bacteremia-associated E. coliO-serotypes from a collection of 860 blood isolates collected in the USand EU in the period 2011-2013. Indicated is the relative O-serotypedistribution of the samples. Bacteremia in ≥60 years old O-serotypeUS/EU 2011-2013 (n = 860) 25 19.2 2 8.8 6 8.3 1 7.8 75 3.3 4 2.8 16 2.718 2.7 15 2.3 8 2.0 153 1.6 73 1.6

Example 11: Induction of Functional Antibody Responses

The functionality of the antibodies raised after vaccination withmonovalent and tetravalent vaccine formulations described above wasinvestigated with an in vitro opsonophagocytic killing (OPK) assay. Thistype of assay has been accepted as a correlate of protection for theconjugate vaccine against Streptococcus pneumoniae (PREVNAR?). The OPKassay measures the ability of serum to facilitate opsonophagocytosis andkilling of different E. coli serotypes. In 96-well plates, defineddilutions of the sample sera were incubated, in each well, with:bacteria from one of the four vaccine-specific E. coli serotypes, adefined amount of HL60 cells, and baby rabbit complement. Afterincubation, a proportion of the mixture was spotted onto tryptic soyagar (TSA) and the number of bacterial colonies was counted. The abilityof the antibodies to bind the bacterial cells and activate deposition ofthe complement and mediate uptake and killing of the bacteria by HL60cells was expressed as opsonic titer. The opsonic titer or opsonizationindex (O1) corresponds to the dilution of the sera killing 50% of thebacterial cells. Opsonic indices for pre and post-immune sera areprovided. A >than 4-fold increase of OI from pre- to post-immune isconsidered significant. OPK assays for three serotypes O2, O6Glc andO25B were established.

Functionality of Antibody Responses Induced by Monovalent Vaccines

To assess the functional activity of vaccine-induced antibody responsesof O25B, O1A, O2 and O6Glc bioconjugates, sera from vaccinated rats wereanalyzed using opsonophagocytic killing (OPK) assays, which measure invitro complement- and antibody-dependent phagocytosis and killing ofbacteria, e.g., E. coli. E. coli was pre-opsonized with dilutions ofserum from vaccinated rats, incubated with complement and phagocytes(differentiated HL60 cells), and the colony forming units (CFUs) weredetermined. Subsequently, the maximum % killing and Opsonization Indices(OI: serum dilution killing of 50% of E. coli) were calculated. E. coliselected for OPK testing were OC 24453 (serotype O2), OC 24781 (serotypeO6Glc) and OC 24176 (serotype O25B). As shown in FIGS. 29A-29C, a robustfunctional immune response to O2-EPA (FIG. 29A), O6Glc-EPA (FIG. 29B)and O25B-EPA (FIG. 29C) was observed.

The data demonstrate that the vaccine components described herein induceantibody responses against E. coli serotypes from which O-antigens areincluded in the vaccine, and that such antibody responses are functionalin killing E. coli from these serotypes.

Functionality of Antibody Responses Induced by a Tetravalent Vaccine

Table 12 shows the total O1 titers for the O-antigens O2, O6Glc and O25Bfrom animals immunized with the tetravalent vaccine with either 0.4 or 4gtg per O-antigen. The titers were determined in two separateexperiments. The 0.4 μg dose induced significant OIs in all animals forthe O2 and O6Glc serotypes. For O25B, 3/8 animals showed a significantincrease in OI following immunization with the 0.4 μg dose. Compared tothe 0.4 μg dose, the 4 μg dose induced lower OI increases for O2 in allanimals. 3/8 animals showed O1 increases when the sera from the 4 μgdose group were tested on O25B E. coli. The data confirm that atetravalent vaccine is able to elicit O-antigen-specific opsonicantibodies against O2, O6Glc and O25B.

The data demonstrate that the vaccine components described herein induceantibody responses against E. coli serotypes from which O-antigens areincluded in the vaccine, and that such antibody responses are functionalin killing E. coli from these serotypes.

TABLE 12 OIs against E. coli O2, O6 and O25. OIs for individualpre-vaccination and post 3 vaccination sera from two separateexperiments are shown for all animals. Tetrayalent-EPA Rat SerumOpsonization indices (OI) O 2 E.coli O 6 E.coli 0.4 μg Dose 4 μg Dose0.4 μg Dose Animal No. Exp. 1 Exp. 2 Exp. 1 Exp. 2 Exp. 1 Exp. 2 1:Pre-acc

7 5 0 17

Post vacc >16364    1′476    2

3 32 202  22

2: Pre-acc 21  11  11  20 11 90 Post vacc 11′148    >16384    150  120436  47

3: Pre-acc 8

0 0  0  5 Post vacc 11′073    >16384    4

1

88 37 4: Pre-acc 5 5 5 8 23 17 Post vacc >16384    63 

7 45 108  116  5: Pre-acc 7 0 0 4 30  8 Post vacc 10′413    7′050   105  108 >16,384    12′672    6: Pre-acc 8 0 8 7 2

9 164  Post vacc 68  34  24  17 1′725   1′475   7: Pre-acc 9

6 8 18 21 Post vacc >16384    >183

4 10

2 1′24

1′8

3 8: Pre-acc 4 6 6 5 2

22 Post vacc 5′05

4′201    3

25 6′

0 3′82

Pre-vacc Av 8 5 5 8 53 42 Post-vacc AV 10′867    7′747    103  57 3′34

3′

6 Tetrayalent-EPA Rat Serum Opsonization indices (OI) O 6 E.coli O 25E.coli 4 μg Dose 0.4 μg Dose 4 μg Dose Animal No. Exp. 1 Exp. 2 Exp. 1Exp. 2 Exp. 1 Exp. 2 1: Pre-acc 6 18  2′404    2′082    0 0 Post vacc2′04

2′

21 1′847    1′578   

0 2: Pre-acc 0 0 0 0 0 0 Post vacc 10′2

2 11′460    0 0 4 0 3: Pre-acc 0 0 0 0 0 0 Post vacc 7′958    8′5

7 6 0 355  1

7 4: Pre-acc 0 0 0 0 0 0 Post vacc 2′1

9 4′4

8 0 0 70  2

5: Pre-acc 8 7 0 0 0 0 Post vacc 3′107    7′584    0 0 105  89  6:Pre-acc 5 0 26

154  0 0 Post vacc

40 5

6 0 0 0 0 7: Pre-acc 22  5 0 0 0 0 Post vacc 180  143  1′130    630 

8 8: Pre-acc 0 0 0 0 0 0 Post vacc 2

8 656  3′336    1′986    0 0 Pre-vacc Av 5 3 334  280  0 0 Post-vacc AV3′318    4′578    790 

24 6

37 

indicates data missing or illegible when filed

The maximum % killing and Opsonization Indices (OI: serum dilutionkilling of 50% of E. coli) were calculated. E. coli selected for OPKtesting were OC 24453 (serotype O2), OC 24781 (serotype O6Glc) and OC24176 (serotype O25B). Robust functional immune response to O2-EPA,O6Glc-EPA and O25B-EPA was observed.

Example 12: Evaluation of a Candidate Vaccine Against UropathogenicEscherichia coli in Women with a Clinical History of Recurrent UrinaryTract Infection (RUTI)

An E. coli bioconjugate vaccine is used in a Phase I clinical study. Thevaccine comprises four bioconjugates in a saline buffer solution. Thefour bioconjugates are: (i) E. coli O1A conjugated to EPA carrierprotein, (ii) E. coli O2 conjugated to EPA carrier protein, (iii) E.coli O6Glc conjugated to EPA carrier protein, and (iv) E. coli O25Bconjugated to EPA carrier protein.

The study population includes 194 healthy females, aged ≥18 to 70 yearsold, with a history of recurrent urinary tract infection (RUTI), definedas ≥3 independent episodes in the previous 12 months or ≥2 episodes inthe last 6 months. At least one of the urinary tract infection (UTI)episodes was caused by E. coli (as single pathogen or part ofpolymicrobial infection) and the cause was culture-confirmed anddocumented. For purposes of the study, UTI is defined by the presence ofat least one specified UTI symptom (dysuria, urgency, frequency, flankpain, bladder tenderness, suprapubic pain, fever, nausea, vomiting)along with a bacterial count (CFU) of ≥10³ CFU/mi uropathogen inmid-stream urine.

The study includes two arms: (i) candidate vaccine and (ii) placebo. Thestudy is a staggered, randomized, single blind, placebo-controlledmulti-center study in healthy women with history of RUTI.

The estimated enrollment period for the study is 4 months, with afollow-up duration period of nine months for each subject.

The objective of the study is to assess the safety, immunogenicity, andefficacy of the E. coli bioconjugate vaccine.

Study Design

Subjects are followed for 9 months after injection, and only injectedsubjects are followed throughout the study period. Subjects attend atotal of 5 scheduled visits: screening (first visit), day 1 (secondvisit), day 7, day 30, and day 270. Subjects receive 4 follow-up phonecalls, on day 2, day 90, day 150, and day 210.

Any unscheduled visits due to occurrence of UTI include standard of carewith harmonized treatment options. Urine and blood (if possible) arecollected for diagnostic and serotyping purposes. Unsolicited adverseevents (AE) and severe adverse events (SAEs) are recorded along thestudy duration whereas solicited AE are recorded for 7 days afterinjection.

At each visit a new diary card is given to the subject and the previousone is discussed.

Dosing and Administration

At first visit, eligible subjects that have provided informed consentare screened and compliance for inclusion/exclusion criteria areconfirmed. Blood is drawn and urine is collected.

At visit 2 (day 1), each subject will receive one intramuscularinjection of 0.5 ml of solution (vaccine candidate or placebo) in thedeltoid muscle. The reduced dose of the candidate vaccine will contain 1μg of each polysaccharide (total 4 μg polysaccharide). The target doseof the candidate vaccine will contain 4 μg of each polysaccharide (total16 μg polysaccharide).

Objectives

The primary objective is to assess the occurrence, intensity,relationship, and duration of solicited and unsolicited adverse events(AE) and serious adverse events (SAE) post-injection of candidatevaccine compared to the placebo group throughout the study period.

The secondary objectives are (i) to compare the change in hematologicaland biochemical safety endpoints prior to injection (at the initialscreening and day 1) and post injection (at day 7 and day 30) ofcandidate vaccine compared to the placebo group; (ii) evaluate theimmune-response of candidate vaccine between baseline (day 1) and postinjection (day 30 and day 270); (iii) compare the number of symptomaticurinary tract infection (UTI) episodes caused by the E. colivaccine-serotypes between the two arms, injected with candidate-vaccineor placebo during the whole study period as most relevant efficacyendpoint; (iv) assess the rate of occurrence of vaccine-serotypespecific E. coli UTI in vaccine group compared to the placebo groupalong the study duration; and (v) assess the intensity and duration ofclinical symptoms of vaccine-serotype specific E. coli UTI in vaccinegroup compared to the placebo group along the study duration.

The exploratory objectives are (i) to compare the rate of UTI occurrencecaused by any E. coli serotype in the vaccine group compared to theplacebo group throughout the study period; and (ii) to compare the rateof UTI occurrence caused by any pathogens in the vaccine group comparedto the placebo group throughout the study period.

Inclusion Criteria

Inclusion criteria for the study are as follows: (i) female subjectswith a history of recurrent UTI, which is defined as: ≥3 UTI independentepisodes in the previous 12 months or ≥2 UTI episodes in the last 6months; at least one UTI during the last 5 years was caused by E. coli(as single pathogen or part of polymicrobial infection) and wasculture-confirmed and documented; (ii) Age ≥18 and ≤70 years; (iii)subjects in a healthy state without ongoing or suspected symptomatic UTIat the screening visit and at injection day (visit 2); (iv) general goodhealth, without clinically significant medical history, physicalexamination findings or clinical laboratory abnormalities per clinicaljudgment of the investigator; and (v) willingness to participate in thestudy after all aspects of the protocol have been explained and fullyunderstood, and written informed consent form obtained.

Exclusion Criteria

Exclusion criteria for the study are as follows: (i) history of morethan 10 recurrent UTIs in the year before the screening visit; (ii) useof any short-term urinary catheter within 7 days prior to screening;(iii) use of any permanent catheter within 30 days prior to screening;(iv) history of any unresolved urinary tract diseases/abnormalities; (v)evidence of impaired immune function; (vi) significant cardiovascular,liver, renal diseases and/or insufficiency; (vii) uncontrolled diabetesmellitus; (viii) significant abnormalities in screening results forhematology, serum chemistry or urinalysis; (ix) positive test for HIV,and/or evidence of HBV or HCV; (x) BMI >34; (xi) previous immunestimulatory therapy for UTI prevention (such as Urovaxom®, Strovac® orUrovac®) in the last 3 months, or planned use during the study period;(xii) current use of any medication known to affect immune function(e.g. corticosteroids ≥0.5 mg/kg BW/day); (xiii) use of UTI-relatedvaginal estrogen treatment newly started less than 6 months beforeinjection and continuing during the study or planned start during theactive study period; (xiv) use of any antibiotic therapy within 1 weekpreceding injection; (xv) planned use of post-coital antibiotics for UTIprevention during study period; (xvi) any vaccination planned within 30days before and 30 days after injection; (xvii) participation in otherclinical trials in the 60 days preceding enrolment and for the durationof the study; (xviii) previous treatment with immunoglobulins or bloodproducts in the 3 months preceding the injection; (xix) knownhypersensitivity to any component of the vaccine; (xx) presence of asignificant medical or psychiatric condition that in the opinion of theinvestigator precludes participation in the study; (xxi) acute illnessat the time of injection; (xxii) women of child bearing potential whoeither have a positive pregnancy test or refuse to use an effectivecontraception; (xxiii) women who are lactating at any time throughoutthe study period; (xxiv) subjects with an elective surgicalintervention, planned during the study period; and (xxv) any othersignificant finding that in the opinion of the Investigator wouldincrease the risk of having an adverse outcome from participating in thestudy.

Statistical Methods and Analysis

Descriptive statistics (n, mean, standard deviation, median and rangesfor continuous variables, frequencies and percentages for categoricalvariables) are provided by treatment group and/or visit, whereapplicable. All data are listed by subject, treatment group and, whereapplicable, visit. All subjects from Group B receiving placebo arecombined to form the placebo treatment group.

Example 13: Phase I Clinical Study Results

This Example presents certain results of the Interim Analysis of thePhase I clinical study described in Example 12.

12.1: Safety

Occurrence of adverse events and severe adverse events were comparablebetween the placebo and vaccinated groups. Ten severe adverse eventswere reported, and none were related to the study drug.

12.2: Immunogenicity

To assess the immunogenicity of the vaccine components, sera from womenparticipating in the clinical study were obtained and analyzed by ELISAto quantify IgG against the four different O-antigens included in thetetravalent vaccine (E. coli O1, E. coli O2, E. coli O6, and E. coliO25B).

Sera from vaccinated women were incubated in plates coated with O1A, O2,O6Glc and O25B-LPS and EPA. Subsequently, plates were incubated withHRP-labeled secondary antibody (anti-human IgG). Bound antibodies weredetected with TMB substrate and absorbance was measured. EC50 valueswere calculated by 4PL fitting.

As shown in FIG. 30, a robust immune response to O1A-EPA, O2-EPA,O6Glc-EPA and O25B-EPA occurred in the majority of the vaccinated women.

These data demonstrate immunogenicity of each component of thetetravalent vaccine.

12.3: Functional Antibody Response

OPK assays, which measure in vitro complement- and antibody-dependentphagocytosis and killing of E. coli bacteria, were used to assess thefunctional antibody response of women participating in the clinicalstudy.

Sera was collected from study participants. E. coli was pre-opsonizedwith dilutions of serum from the vaccinated women, incubated withcomplement and phagocytes (differentiated HL60 cells), and the remainingcolony forming units (CFUs) was determined. Subsequently, the maximumpercent killing and Opsonization Indices (01: serum dilution killing of50% of E. coli) were calculated. E. coli selected for OPK testing wereOC 24452 (serotype O1A), OC 24453 (serotype O2), OC 24454 (serotypeO6Glc), and OC 24176 (serotype O25B).

As shown in FIGS. 31A-31D, a robust functional immune response toO1A-EPA (FIG. 31A), O2-EPA (FIG. 31B), O6Glc-EPA (FIG. 31C), andO25B-EPA (FIG. 31D) was observed.

These data demonstrate that each component of the tetravalent vaccineinduces a serotype-specific antibody response, and that such antibodyresponses are functional in killing E. coli from these serotypes. Thus,the vaccine compositions described herein are functional in humans.

12.4: Immunization with a Tetravalent O-Antigen Conjugate ComprisingO25B-EPA Elicits O25A/O25B Cross-Reactive IgG Antibodies In Humans

To determine the level of cross-reactivity of vaccine-induced serum IgGantibodies toward the two known E. coli O25 serosubtypes, O25A and O25B,serial dilutions of serum derived from vaccinated subjects wereincubated with purified O25A LPS, O25B LPS, or intact bacterial cells,and tested by ELISA.

As shown in FIG. 32, similar EC₅₀ values were observed when reactivitytowards O25A LPS (black bars) and O25B LPS (grey bars) thirty days postvaccination was tested. Overall the data suggest that the O25Bbioconjugate works well for O25B and for O25A, but in most cases/testedsubjects O25B works slightly better in terms of antibody response forO25B compared to O25A. This result demonstrates that with the occurrenceof some natural variation, the tetravalent vaccine induces antibodiesthat recognize both O25A and O25B LPS. To test whether the same was truefor whole bacterial cells and multiple O25A/O25B strains, reactivitytowards a set of clinical O25A or O25B isolates derived from eitherblood or urine was also tested. In this case a serotype O75 strain, aserotype not represented in the tetravalent vaccine, was used as anegative control (dotted grey line in FIG. 33). As demonstrated in FIG.33, vaccine-induced serum IgG antibodies showed a strong response towardeach of the individual O25 strains. Although strain-to-strain variationwas apparent, reactivity toward the O25A (black lines) and O25B strains(grey lines) was observed. These data demonstrate that the O25B vaccinecomponent of the tetravalent vaccine elicits antibodies that recognizeboth O25A and O25B purified LPS and E. coli O25A and O25B strains.

Tables 13 and 14, below, provide details of certain strains andplasmids, respectively, used in the foregoing examples.

TABLE 13 Strains Name Genotype Description upec032 wt O1A clinicalisolate from GVXN epidemiology study upec436 wt O25A clinical isolatefrom GVXN epidemiology study upec138 wt O25B clinical isolate from GVXNepidemiology study upec116 wt O2 clinical isolate from GVXN epidemiologystudy upec163 wt O25B clinical isolate from GVXN epidemiology studyupec177 wt O25B clinical isolate from GVXN epidemiology study W3110 F-,λ⁻, K-12 laboratory strain used IN(rrnD-rrnE)1, rph-1 for productionstrain synthesis, CGSC#: 4474 CCUG25 wt O2 isolate obtained from theculture collection, University of Göteborg (CCUG), Sweden (see Jansson,et al., (1987) Carbohydrate research 161, 273-27) CCUG11309 wt O6isolate from the CCUG, OPS with branching Glc (see Jann, et al., (1994)Carbohydrate research 263, 217-225) CCUG11311 wt O6 isolate from theCCUG, OPS with branching GlcNAc (see Jann, et al., (1994) Carbohydrateresearch 263, 217-225)

TABLE 14 Plasmids Name Description Remarks pGVXN150 pBR322 basedexpression See Ihssen, et al., (2010) plasmid of genetically Microbialcell factories 9, 61 detoxified EPA-his6 encoding 2 glycosylatiion sitespGVXN659 pBR322 based expression pGVXN150 was modified to plasmid ofgenetically encode additional N and C detoxified EPA encoding 4 terminalglycosylation sites glycosylatiion sites pGVXN1076 pGVXN659 ΔampR::kanRpGVXN579 pMAL-p2X based vector Allows quick bioconjugates for expressionof MBP with purification avoiding a a C terminal flexible linker, histagfollowed by 3 glycosylation site sequences and a myc epitope pGVXN114pEXT21 based expression See Ihssen, et al., (2010) plasmid for PglB withan Microbial cell factories 9, 61 HA tag pGVXN939 pEXT21 basedexpression plasmid for PglB with an HA tag, codon optimized pGVXN970pEXT21 based expression plasmid for PglB without tag, codon optimizedpGVXN539 pACT3 based expression replaced chloramphenicol plasmid forgenetically resistance by kanamycin detoxified, codon usage frompGVXN161 (oligos: optimized, histagged EPA #1399/#1400) encoding 2glycosylation sites as pGVXN150 pGVXN161 pKD4 See Datsenko and Wanner,(2000) Proc Natl Acad Sci USA 97, 6640-6645 pGVXN112 pACT3 basedexpression plasmid for PglB with an HA tag

TABLE 15 Sequences Description SEQUENCE SEQ ID NO. rmlB (upec138)GTGAAGATACTTGTTACTGGTGGCGCAGGATTTATTGGTTCTGCT  1GTTGTTCGTCACATAATAAATAATACGCAAGATAGTGTTGTTAATGTCGATAAATTAACATACGCCGGAAACCTGGAATCACTTGCAGATGTTTCTGATTCTGAACGCTATTTCTTTGAACATGCGGATATTTGTGATGCAGCTGCAATGGCACGGATTTTTGCTCAGCATCAGCCGGATGCAGTGATGCACCTGGCAGCTGAAAGCCATGTTGACCGTTCAATTACAGGCCCTGCGGCATTTATTGAAACCAATATTGTGGGTACTTATGTCCTTTTAGAAGCGGCTCGGAATTATTGGTCTGGTCTGGATGATGAAAAGAAAAAAAACTTCCGTTTTCATCATATTTCTACTGATGAGGTGTATGGTGACTTACCCCATCCGGATGAAGTAAATAGCAATGAAACGTTGCCGCTATTTACGGAAACGACAGCATACGCGCCAAGTAGTCCATATTCTGCTTCTAAAGCTTCCAGCGATCATTTGGTTCGCGCATGGAAACGTACTTATGGTTTACCGACCATTGTGACTAATTGCTCGAACAACTATGGTCCTTATCATTTCCCGGAAAAGCTTATTCCACTGGTTATTCTTAATTCACTGGAAGGTAAGGCATTACCTATTTATGGCAAAGGAGATCAGATCCGCGACTGGTTGTATGTAGAGGATCATGCTCGAGCGTTATATACCGTCGTAACCGAAGGTAAAGCGGGCGAAACTTATAACATTGGTGGACACAACGAAAAGAAAAACATCGACGTAGTGTTCACTATTTGTGATTTGTTGGATGAGATAGTCCCGAAAGAGAAATCTTACCGCGAGCAAATTACTTATGTTACCGATCGTCCGGGACACGATCGCCGTTATGCGATTGATGCTGAGAAGATTGGTCGCGAATTGGGATGGAAACCACAGGAAACGTTTGAGAGTGGGATTCGTAAAACGGTGGAATGGTACCTGTCCAATACAAAATGGGTTGATAATGTGAAAAGTGGTGCCTATCAATCGTGGATTGAACAGAACTATGAGGGCCGC CAGTAA rmlD (upec138)ATGAATATCCTCCTTTTTGGCAAAACAGGGCAGGTAGGTTGGGAA  2CTACAGCGTGCTCTGGCACCTCTGGGTAATTTGATTGCTCTTGATGTTCACTCCACTGATTACTGTGGTGATTTTAGTAATCCTGAAGGTGTAGCTGAAACCGTAAGAAGCATTCGGCCTGATATTATTGTCAACGCAGCCGCTCACACCGCAGTAGACAAAGCAGAATCAGAACCGAAGTTTGCACAATTACTGAACGCGACGAGTGTCGAAGCGATCGCGAAAGCAGCCAATGAAGTCGGCGCCTGGGTTATTCACTACTCTACTGACTACGTATTTCCGGGGACCGGTGAAATACCATGGCAGGAGGAGGATGCAACCGCACCGCTAAATGTTTACGGTGAAACCAAGTTAGCGGGAGAAAAAGCATTACAAGAGCATTGTGCGAAGCACCTTATTTTCCGGACCAGCTGGGTCTATGCAGGTAAAGGAAATAACTTCGCCAAAACAATGTTGCGTCTGGCAAAAGAGCGTGAAGAATTAGCCGTTATTAATGATCAGTTTGGTGCGCCAACTGGCGCAGAGTTACTGGCTGATTGTACGGCACATGCTATTCGTGTGGCACTGAATAAACCGGAAGTCGCAGGCTTGTACCATCTGGTAGCTAGTGGTACCACAACGTGGCACGATTATGCTGCGCTGGTTTTTGAAGAGGCGCGCAAAGCAGGCATTCCCCTTGCACTCAACAAGCTCAACGCAGTACCAACAACAGCCTATCCTACACCAGCTCGTCGTCCACATAACTCTCGCCTTAATACAGAAAAATTTCAGCAGAACTTTGCGCTTGTCTTGCCTGACTGGCAGGTTGGCGTGAAACGAATGCTTAACGAATTATTTACGACTACAGCAATTTAA rmlA (upec138)ATGAAAACGCGTAAAGGTATTATTTTGGCGGGTGGTTCTGGTACT  3CGTCTTTATCCTGTGACGATGGCCGTCAGTAAACAGCTGTTACCGATTTATGATAAACCGATGATCTATTACCCGCTCTCTACACTGATGTTAGCGGGTATTCGCGATATTCTGATTATCAGTACACCACAGGATACTCCTCGTTTTCAACAACTGCTGGGTGACGGGAGCCAGTGGGGCCTGAATCTTCAGTACAAAGTGCAACCGAGTCCGGATGGTCTTGCGCAGGCGTTTATTATCGGTGAAGAGTTTATTGGTGGTGATGATTGTGCTTTGGTACTTGGTGATAATATCTTCTACGGCCACGACCTGCCGAAGTTAATGGACGTAGCTGTTAACAAAGAAAGTGGTGCAACGGTATTTGCCTATCACGTTAATGATCCTGAACGTTATGGTGTCGTGGAGTTTGATAATAACGGTACTGCAATTAGCCTGGAAGAAAAACCGCTGGAACCAAAAAGTAACTATGCGGTTACTGGGCTTTATTTCTATGACAATGACGTTGTGGAAATGGCGAAAAACCTTAAGCCTTCTGCCCGAGGTGAACTGGAAATTACCGATATTAACCGTATTTATATGGAACAAGGACGTTTGTCTGTCGCTATGATGGGGCGTGGCTATGCATGGCTGGATACAGGGACGCATCAAAGTCTTATTGAAGCAAGCAACTTCATTGCCACCATTGAAGAGCGCCAGGGACTAAAGGTTTCCTGTCCGGAAGAAATTGCTTATCGTAAAGGGTTTATTGATGCTGAGCAGGTAAAAGTATTAGCCGAACCGTTGAAGAAAAATGCTTATGGTCAGTATCTG CTCAAAATGATTAAAGGTTATTAArmlC (upec138) ATGAACGTAATTAAAACTGAAATTCCTGATGTGCTGATTTTTGAA  4CCAAAAGTTTTTGGGGATGAACGTGGCTTCTTTTTTGAGAGTTTTAATCAGAGGATTTTTGAAGAAGCAGTAGGTCGTAAGGTTGAGTTTGTTCAGGATAACCATTCTAAGTCCAGTAAAGGTGTTTTACGTGGTCTTCATTATCAGTTAGAACCTTATGCTCAAGGAAAACTGGTGCGCTGTGTTGTTGGCGAGGTTTTTGATGTTGCGGTTGATATTCGTAAATCGTCACCTACATTTGGGAAATGGGTTGGGGTGAATTTGTCTGCTGAGAATAAGCGTCAGTTGTGGATTCCTGAGGGATTTGCACATGGTTTTTTGGTGCTGAGTGATTTAGCAGAAGTTTTATATAAAACGAATCAATATTATGCTCCATCACATGAAAAAAATATTATATGGAATGACCTCTTGCTTAATATTAAATGGCCGAGCACAGCACTGATCACTCTGTCTGATAAGGATGCAAATGGGGAAAGATTTGAACTAAGTGAGTTT TGA wzx (upec138)ATGTCTCTCTTAAAACATAGTATATGGAATGTTGCGGGCTACTTT  5ATACCAACATTAATTGCAATTCCCGCCTTTGGATTAATTGCGAGGAAAATTGGTGTAGAACTATTTGGTTTGTATACGTTAGCAATGATTTTTATAGGGTATGCAAGTATATTTGATGCTGGGTTAACAAGAGCTGTTGTGCGTGAAATAGCATTACTAAAAAACAGAGTGGACGATTGTAATACGATAATAGTAACTTCTATTATCGCTGTGATATTTTTAGGGTTTATCGGAGGCGGGGGAGTGTTTCTGCTTAAAGGCGATATTATTGAACTGTTAAATATCTCACCAATATATTACGCCGATTCGATAAAGTCTCTAGTATTATTATCATCTCTGATACCTGTATTCTTAGTCACGCAAATACTATTAGCAGAGCTTGAGGGTCGGGAATATTTTGGGATTCTAAATATACAAAAAAGTGTAGGGAATTCTTTAATTGCAGGGTTACCTGCATTATTTGTTTTAATTAATCAAACGCTTTTTTCTGCAATTATTGGTGTAGCGATTGCAAGAGTTATATGCTTGTGGTTAAGCTACATTATGAGCAGGGAAAGAATAACTATCGATATCTCATTTTTTTCAATAACTGTTTTAAAGCGGTTATTTAGATATGGCGGGTGGGTAACTATAAGTAACATAATATCTCCTATATTAGCGAGTATGGATAGATTTATTCTATCCCATATCCAGGGAGCATCAAAAATATCATTCTATACAGTCCCTAATGAGCTGGTAACTAGGCTTGGAATAGTTCCAGGCTCTCTTGGGAAAGCTGTTTTTCCAAAATTAAGTCATGCAAGGAATTTTACAGCGTCATATGCAGAGCAAAAAAAAGCTTATATATTAATGACTGTCATTGTAATGCCTTTGGTTTTATTTGTATATTATTACGCAAAGTTTATTTTAACATTGTGGATGGGGGCTGAGTATGCAGGGATTTCGGTCGAAATATTACGGATTATGCTTATAGGGTATATTTTTAACTGTTATTCACAAATCTCTTTTGCCAACATACAGGCCTTTGGAAAAGCAAAATACACTGCATACATCCATATGATGGAATTTATTCCTTATTTGATAATGTTATATATAATTTCAAAGGAATATGGGGTTATTGGTGTTGCGTGGTTATGGACAATTCGAGTAATAATTGATTTTTTGATGCTTTTATATATGAGTTATCGTTGTAATAATCTTATGAAAAAAGGGTAG wekA (upec138)ATGATATATATTGTGGTATTAAATTGGAATGGGGCTATAGATACC  6ATTAATTGTGTTAAAAGTTTAATGGATTTAAATGTTAGCGATTATAAAATTATCATTGTTGATAACTGTTCTATGGATAACTCATATGATACTATAAAAGAAAATCTTAATTCATTATATATTGCTGATAAAAGTATCATTGAGGTGAAGTATGAGGATAGAAATAAATATAAAACCTTAGAAAACGATAAAATCATATTAATACAATCTCCGCAAAATAATGGGTACGCAAGTGGTAATAATATTGGCATAGAGTTCGCTCTTAATCAGGAGAATATGAAATACGTCTGGGTTCTGAATAATGATACTGAAGTGGATAAAGAGGCTTTAACTCATTTAATTAGTAAATGTGATTCAGATAAAAGTATAGGGATTTGCGGTTCTCGTTTAGTCTATTTTGCCGACAGAGAGATGCAGCAAGGACTAGGTGGGGTGCATAACAAATGGTTATGCACTACAAAAAATTATGAAATGGGAAGATTAGTTTCCAAAAAATATGATGATGAAGTCATTAGTAATGATATAGATTATATAATTGGCGCATCGATGTTTTTCTCTAGAGAATGTTTGGAAACAGTTGGATTGATGAATGAAGAATATTTTTTATACTATGAAGAGTTAGATATTTGCCTCAGAGCAAAAGCAAAGAACTTTAAATTAGGTATTTGCTCAGAAAGTTTGGTTTATCATAAAATAGGTGCAAGTACTGATGGGGGAAAGAGCATGATGGCTGATCTTTGCTCAATAAAAAATAGGCTGGTCATTACAGAAAGGTTTTATCCCCAATATTATTGGACGGTATGGTTGTCACTTTTTGTTGTAGCATTTAACCGTGCTAGAAGAGGTGAGTTTAATAAGATGAAAAGATGTTTGAATGTTATGTTTAACTTCAAACGAAAC AAAGGTAGCAAATGCCATTAGwekB (upec138) ATGAAAGTGGCTTTTTTATCTGCTTATGATCCACTATCTACATCC  7AGTTGGTCTGGCACACCTTATTATATGCTAAAGGCATTATCGAAGAGAAATATTTCCATTGAAATATTAGGACCGGTAAATAGCTATATGATATACATGTTAAAAGTATATAAATTAATATTAAGGTGTTTCGGAAAAGAATATGATTATAGTCATTCGAAGTTGCTTTCCAGGTATTACGGTAGAATATTCGGTAGGAAATTAAAAAAAATTGATGGTTTGGATTTTATTATCGCACCTGCAGGTTCCTCACAAATTGCTTTTTTAAAAACAACCATACCAATAATATATCTATCGGATACAACATATGATCAATTAAAAAGCTATTATCCGAATTTAAATAAAAAAACAATTATAAATGATGAGGATGCAAGTTTAATCGAACGCAAGGCTATTGAAAAAGCAACAGTAGTATCTTTCCCATCTAAATGGGCAATGGATTTTTGCAGGAATTATTACAGATTAGATTTTGATAAATTAGTTGAAATACCATGGGGGGCTAATTTATTTGATGATATTCACTTTGCTAATAAAAATATAATTCAAAAGAATAGTTATACTTGTCTTTTCTTGGGAGTTGATTGGGAAAGAAAAGGTGGGAAAACAGCCTTGAAAGCAATTGAATATGTAAGGCAGTTATATGGGATCGATGTTAGACTAAAAATTTGTGGATGTACTCCGAATCAAAAGATTTTACCTACTTGGGTTGAATTAATTGATAAAGTAGATAAAAATAACGTTGACGAATATCAGAAATTCATCGATGTGTTATCTAACGCTGATATACTTCTTTTACCAACCATTGCTGAATGTTATGGAATGGTATTTTGTGAAGCTGCTGCTTTTGGATTGCCTGTTGTCGCTACAGATACAGGTGGAGTCAGTTCTATAGTTATCAACGAAAGGACGGGGATATTAATTAAAGACCCGTTAGACTATAAGCACTTTGGAAATGCAATTCATAAAATAATTAGTTCCGTAGAGACTTATCAAAACTACTCCCAAAACGCAAGAATTAGATATAATAATATATTGCATTGGGACAATTGGGCTAAAAAGATAATTGAGATTATGTATGAG CATAAGAATAGAAGAATCAAATAGwzy (upec138) ATGAGCATAAGAATAGAAGAATCAAATAGCACAAAAAGAATTATA  8TGTTTATTTATACTTTTTCTTGTTTTCCCTGATTTTTTGTTTTATACATTAGGGGTTGATAATTTTAGCATTTCAACGATAATCTCAATTACATTGCTTTTTGTTTTTTTAAGAGCTAAAAATATTTGCAAAGATAATTTTCTAATAATAGTAGCGTTATTCATATTGTTGTGTTTTAACTGTTTGTTAAGTATGCTATTTAATATTGAACAGGCTTTAACATTTAAAGTTGTACTTTCAATATATAGCATCTTAATAATGGCATACGTCTCCTCTTGTTATGCACAGACGTTGTGGTTATGTTCTGAAGAAATACTTAAGAGATCCGTCTTTTATTTGTTCGCATTTCTTTGCCTTATTGGCATTATAAGTATTCTTTTACAGAAGACTGAGATTATACATGATAAAAGTATGATTCTTTTTCCTGAACCATCAGCATTTGCATTGGTTTTTATACCTATCTTTTCATTTTGTTTATACTATACAAGAGGGGGGGGGCTACTATTGCTCTATATATTATCTTTGGGTATTGCGTTAGGTATCCAGAATTTAACAATGTTGGTAGGCATTGTGATTAGTGTTTTTGTGATGAAAAAAATAACTATAAGGCAAACTATTGTTATACTTTTGGGGGCATGGATTTTTTCCATGATATTAAGTGATTTAGACATTTCTTACTATACATCGCGGCTTGATTTTAAAAATACTACGAACCTATCAGTGCTTGTATATCTTTCAGGAATTGAAAGAGCTTTCTTGAATTTTATTACAAGTTATGGTCTTGGTATTGGTTTTCAACAAATGGGAGTGAATGGGGAGATAGGAATATATCAACAAATTTTAGCTGAACTTGATGCCCCTATGTTAAATATATACGATGGCTCATTTATTTCTTCTAAGTTTTCTATTTTTTTTATTTTTCCCGATTTTATCTGCGTTTCAAAAAAAGTAAGAGATATTCACCGCAGTATATTTTAGCATATAGCTTCTACATGTGTTTCTTCATCCCTCTTTTTATACGTGGTGCTGGTTATATAAACCCCTATGTGTTTATGTTATTTTCATCAATATTTTTGTGCAAATATCACGCTAAAAATATCTTGATGAAATCTAATGTCCAGATAG CTATATAA wbbJ (upec138)ATGTGCATTAAAAAAAAACTTAAGTTAATTAAACGATATGGCCTT  9TATGGTGGTCTTAGGCTTCTTAAAGATATATTCTTAACAAAATTTTTATTTTGTTCAAATGTTAGGATTATTAGATTTCCATGTTATATTAGAAAAGATGGAAGTGTTAGTTTTGGAAAAGGTTTTACATCAGGTGTAGGATTACGAGTTGATGCATTTATGGATGCCGTAGTTTCCATTGGAGAAAATGTTCAAATTAATGACTATGTTCACATCGCGGCTATTAATAATGTCATTATTGGTAGAGATACATTAATAGCAAGTAAAGTATTTATTAGTGATCATAATCATGGTATTTTTTCTAAATCCGATATCCATAGTTCACCAACTATTATTCCTTCGTCTAGGCCCCTTGAATCTGCACCTGTGTATATTGGAGAGCGTGTGTGGATTGGCGAAAATGTGACAATATTACCAGGTGCGTGTATAGGTAATGGTGTAGTTATTGGCGCAAACAGTGTTGTTCGTGGTGAGATTCCTAATAATGTGATCATTGCTGGTGTTCCAGCTAAAATTGTTAAAAAATATAACTATGAGCGT ATGCAATGGGAAAGAATATAGwbbK (upec138) ATGGGAAAGAATATAGTTGTAATATCGGCTGTTAATTTTACAACC 10GGAGGCCCCTTTACCGTACTAAAAAATGTGCTTACAGCAACTAAAGATAGAGCCGAATGTAAATTTATTGCACTGGTTCATAGCTCTGCTGAACTAATGGAATTATTTCCGTGGGTTGAATTTATAGAGTATCCAGAAGTCAAGTCTTCGTGGGTTAAAAGATTATATTTCGAATATATAACTTGCAATAGATTATCTAAGGTGATTAAGGCAACTCATTGGGTATGCTTACATGATATTACAGCAAATGTTAGTGTACCCTATAGATTTGTTTATTGCCACAATCCTGCACCGTTCTATAAATATTTAAGCTATCGAGATATTATAGGAGAACCTAAATTTTATCTTTTTTATCTTTTTTATGGGCTTTTATACAATATCAATATAAAAAAGAACACAGCAGTTTTTGTTCAGCAGCAGTGGCTAAAAAAAGAATTCGAAAAAAAATATAAGTTAAAGAATGTTGTTGTTAGTCGCCCTGAAGATATTTGCCCTTTTGAAAGTGATGGTTTGGTAAGAAATAATAATAAAAAGGATGTGAGGATATTTTACCCAGCAGTGCCCCGTATATTTAAAAACTTTGAAGTTATCATACGTGCTGCACAAATATTACAAGATAAAAATATTCATTTTTATCTTACTTTTGATGGTACTGAAAATAAGTATGCAAAAAGAATATATAAATTAGCTTCCGAACTGAAAAATGTACATTTCCTCGGTTACCTTAATGCAACCGAGATGGTTAACTTTTATCAAGATTCAGATATTATTTGTTTCCCATCGAAACTAGAAACGTGGGGATTACCATTATCAGAAGCTAAAACATACAAAAAATGGATATTTGCGGCAGACTTACCTTATGCTCATGAAGTTTTATATACTATTCAAAAACTAGATATTTTCCATTTGACGATGAGAAAATACTTGTTCGCTACATATTAGAGTACACAAGTAAAAATATGCATGAAGATATAAAAAATAGTAGGGTGAATTTTAATAATGATGCATTGACTGGTTTTGAACAGTTTATTGAAT ATATCCTCAAGGGGAACTGAwbbL (upec138) ATGATTATGAATAATGATTATTTTCTCTTTCTTAACCCCGATGTA 11TTCATAACCAGTGAAAGTTTGATTAATTATGTTGATTATATAATTAGTAATGATTATAAGTTTAGCACATTATGTCTTTATCGAGATTTTACTAAAAGCAAACATGATTATTCAATACGGAGTTTTCCAACTTTATATGATTTTCTTTGTTCTTTTTTATTGGGGGTGAATAAAAGTAAAATTAAGAAGGAAAATATACTTTCTGATACTGTAGTTGATTGGTGTGCTGGCTCATTTATGCTTATTCATGCTTTAAGTTTCTTAAATGTGAATGGTTTTGATCAAAAATATTTTATGTATTGTGAAGATATTGACCTTTGTATGCGTTTAAAATTAAGTGGAGTAGATCTTTACTATACTCCCCATTTTGATGCTATTCATTATGCGCAGCATGAAAATAGAAGAATATTTACTAAAGCATTTCGATGGCATATAAGGAGTATTACGCGCTACATATTACGGAAACCAATTCTTTCTTATAAAAACTATAGAAAA ATTACATCCGAACTGGTAAAGTGAE. coli GTGAAGATACTTGTTACTGGTGGCGCAGGATTTATTGGTTCTGCT 12 rfb(upec138)GTTGTTCGTCACATAATAAATAATACGCAAGATAGTGTTGTTAAT gene clusterGTCGATAAATTAACATACGCCGGAAACCTGGAATCACTTGCAGATGTTTCTGATTCTGAACGCTATTTCTTTGAACATGCGGATATTTGTGATGCAGCTGCAATGGCACGGATTTTTGCTCAGCATCAGCCGGATGCAGTGATGCACCTGGCAGCTGAAAGCCATGTTGACCGTTCAATTACAGGCCCTGCGGCATTTATTGAAACCAATATTGTGGGTACTTATGTCCTTTTAGAAGCGGCTCGGAATTATTGGTCTGGTCTGGATGATGAAAAGAAAAAAAACTTCCGTTTTCATCATATTTCTACTGATGAGGTGTATGGTGACTTACCCCATCCGGATGAAGTAAATAGCAATGAAACGTTGCCGCTATTTACGGAAACGACAGCATACGCGCCAAGTAGTCCATATTCTGCTTATAAAGCTTCCAGCGATCATTTGGTTCGCGCATGGAAACGTACTTATGGTTTACCGACCATTGTGACTAATTGCTCGAACAACTATGGTCCTTATCATTTCCCGGAAAAGCTTATTCCACTGGTTATTCTTAATTCACTGGAAGGTAAGGCATTACCTATTTATGGCAAAGGAGATCAGATCCGCGACTGGTTGTATGTAGAGGATCATGCTCGAGCGTTATATACCGTCGTAACCGAAGGTAAAGCGGGCGAAACTTATAACATTGGTGGACACAACGAAAAGAAAAACATCGACGTAGTGTTCACTATTTGTGATTTGTTGGATGAGATAGTCCCGAAAGAGAAATCTTACCGCGAGCAAATTACTTATGTTACCGATCGTCCGGGACACGATCGCCGTTATGCGATTGATGCTGAGAAGATTGGTCGCGAATTGGGATGGAAACCACAGGAAACGTTTGAGAGTGGGATTCGTAAAACGGTGGAATGGTACCTGTCCAATACAAAATGGGTTGATAATGTGAAAAGTGGTGCCTATCAATCGTGGATTGAACAGAACTATGAGGGCCGCCAGTAATGAATATCCTCCTTTTTGGCAAAACAGGGCAGGTAGGTTGGGAACTACAGCGTGCTCTGGCACCTCTGGGTAATTTGATTGCTCTTGATGTTCACTCCACTGATTACTGTGGTGATTTTAGTAATCCTGAAGGTGTAGCTGAAACCGTAAGAAGCATTCGGCCTGATATTATTGTCAACGCAGCCGCTCACACCGCAGTAGACAAAGCAGAATCAGAACCGAAGTTTGCACAATTACTGAACGCGACGAGTGTCGAAGCGATCGCGAAAGCAGCCAATGAAGTCGGCGCCTGGGTTATTCACTACTCTACTGACTACGTATTTCCGGGGACCGGTGAAATACCATGGCAGGAGGAGGATGCAACCGCACCGCTAAATGTTTACGGTGAAACCAAGTTAGCGGGAGAAAAAGCATTACAAGAGCATTGTGCGAAGCACCTTATTTTCCGGACCAGCTGGGTCTATGCAGGTAAAGGAAATAACTTCGCCAAAACAATGTTGCGTCTGGCAAAAGAGCGTGAAGAATTAGCCGTTATTAATGATCAGTTTGGTGCGCCAACTGGCGCAGAGTTACTGGCTGATTGTACGGCACATGCTATTCGTGTGGCACTGAATAAACCGGAAGTCGCAGGCTTGTACCATCTGGTAGCTAGTGGTACCACAACGTGGCACGATTATGCTGCGCTGGTTTTTGAAGAGGCGCGCAAAGCAGGCATTCCCCTTGCACTCAACAAGCTCAACGCAGTACCAACAACAGCCTATCCTACACCAGCTCGTCGTCCACATAACTCTCGCCTTAATACAGAAAAATTTCAGCAGAACTTTGCGCTTGTCTTGCCTGACTGGCAGGTTGGCGTGAAACGAATGCTTAACGAATTATTTACGACTACAGCAATTTAATAGTTTTTGCATCTTGTTCGTAATGGTGGAGCAAGATGTATTAAAAGGAATGATGAAATGAAAACGCGTAAAGGTATTATTTTGGCGGGTGGTTCTGGTACTCGTCTTTATCCTGTGACGATGGCCGTCAGTAAACAGCTGTTACCGATTTATGATAAACCGATGATCTATTACCCGCTCTCTACACTGATGTTAGCGGGTATTCGCGATATTCTGATTATCAGTACACCACAGGATACTCCTCGTTTTCAACAACTGCTGGGTGACGGGAGCCAGTGGGGCCTGAATCTTCAGTACAAAGTGCAACCGAGTCCGGATGGTCTTGCGCAGGCGTTTATTATCGGTGAAGAGTTTATTGGTGGTGATGATTGTGCTTTGGTACTTGGTGATAATATCTTCTACGGCCACGACCTGCCGAAGTTAATGGACGTAGCTGTTAACAAAGAAAGTGGTGCAACGGTATTTGCCTATCACGTTAATGATCCTGAACGTTATGGTGTCGTGGAGTTTGATAATAACGGTACTGCAATTAGCCTGGAAGAAAAACCGCTGGAACCAAAAAGTAACTATGCGGTTACTGGGCTTTATTTCTATGACAATGACGTTGTGGAAATGGCGAAAAACCTTAAGCCTTCTGCCCGAGGTGAACTGGAAATTACCGATATTAACCGTATTTATATGGAACAAGGACGTTTGTCTGTCGCTATGATGGGGGCGTGGCTATGCATGGCTGGATACAGGGACGCATCAAAGTCTTATTGAAGCAAGCAACTTCATTGCCACCATTGAAGAGCGCCAGGGACTAAAGGTTTCCTGTCCGGAAGAAATTGCTTATCGTAAAGGGTTTATTGATGCTGAGCAGGTAAAAGTATTAGCCGAACCGTTGAAGAAAAATGCTTATGGTCAGTATCTGCTCAAAATGATTAAAGGTTATTAATAAGATGAACGTAATTAAAACTGAAATTCCTGATGTGCTGATTTTTGAACCAAAAGTTTTTGGGGATGAACGTGGCTTCTTTTTTGAGAGTTTTAATCAGAGGATTTTTGAAGAAGCAGTAGGTCGTAAGGTTGAGTTTGTTCAGGATAACCATTCTAAGTCCAGTAAAGGTGTTTTACGTGGTCTTCATTATCAGTTAGAACCTTATGCTCAAGGAAAACTGGTGCGCTGTGTTGTTGGCGAGGTTTTTGATGTTGCGGTTGATATTCGTAAATCGTCACCTACATTTGGGAAATGGGTTGGGGTGAATTTGTCTGCTGAGAATAAGCGTCAGTTGTGGATTCCTGAGGGATTTGCACATGGTTTTTTGGTGCTGAGTGATTTAGCAGAAGTTTTATATAAAACGAATCAATATTATGCTCCATCACATGAAAAAAATATTATATGGAATGACCTCTTGCTTAATATTAAATGGCCGAGCACAGCACTGATCACTCTGTCTGATAAGGATGCAAATGGGGAAAGATTTGAACTAAGTGAGTTTTGAAATGTCTCTCTTAAAACATAGTATATGGAATGTTGCGGGCTACTTTATACCAACATTAATTGCAATTCCCGCCTTTGGATTAATTGCGAGGAAAATTGGTGTAGAACTATTTGGTTTGTATACGTTAGCAATGATTTTTATAGGGTATGCAAGTATATTTGATGCTGGGTTAACAAGAGCTGTTGTGCGTGAAATAGCATTACTAAAAAACAGAGTGGACGATTGTAATACGATAATAGTAACTTCTATTATCGCTGTGATATTTTTAGGGTTTATCGGAGGCGGGGGAGTGTTTCTGCTTAAAGGCGATATTATTGAACTGTTAAATATCTCACCAATATATTACGCCGATTCGATAAAGTCTCTAGTATTATTATCATCTCTGATACCTGTATTCTTAGTCACGCAAATACTATTAGCAGAGCTTGAGGGTCGGGAATATTTTGGGATTCTAAATATACAAAAAAGTGTAGGGAATTCTTTAATTGCAGGGTTACCTGCATTATTTGTTTTAATTAATCAAACGCTTTTTTCTGCAATTATTGGTGTAGCGATTGCAAGAGTTATATGCTTGTGGTTAAGCTACATTATGAGCAGGGAAAGAATAACTATCGATATCTCATTTTTTTCAATAACTGTTTTAAAGCGGTTATTTAGATATGGCGGGTGGGTAACTATAAGTAACATAATATCTCCTATATTAGCGAGTATGGATAGATTTATTCTATCCCATATCCAGGGAGCATCAAAAATATCATTCTATACAGTCCCTAATGAGCTGGTAACTAGGCTTGGAATAGTTCCAGGCTCTCTTGGGAAAGCTGTTTTTCCAAAATTAAGTCATGCAAGGAATTTTACAGCGTCATATGCAGAGCAAAAAAAAGCTTATATATTAATGACTGTCATTGTAATGCCTTTGGTTTTATTTGTATATTATTACGCAAAGTTTATTTTAACATTGTGGATGGGGGCTGAGTATGCAGGGATTTCGGTCGAAATATTACGGATTATGCTTATAGGGTATATTTTTAACTGTTATTCACAAATCTCTTTTGCCAACATACAGGCCTTTGGAAAAGCAAAATACACTGCATACATCCATATGATGGAATTTATTCCTTATTTGATAATGTTATATATAATTTCAAAGGAATATGGGGTTATTGGTGTTGCGTGGTTATGGACAATTCGAGTAATAATTGATTTTTTGATGCTTTTATATATGAGTTATCGTTGTAATAATCTTATGAAAAAAGGGTAGCCTGATGATATATATTGTGGTATTAAATTGGAAATGGGGCTATAGATACCATTAATTGTGTTAAAAGTTTAATGGATTTAAATGTTAGCGATTATAAAATTATCATTGTTGATAACTGTTCTATGGATAACTCATATGATACTATAAAAGAAAATCTTAATTCATTATATATTGCTGATAAAAGTATCATTGAGGTGAAGTATGAGGATAGAAATAAATATAAAACCTTAGAAAACGATAAAATCATATTAATACAATCTCCGCAAAATAATGGGTACGCAAGTGGTAATAATATTGGCATAGAGTTCGCTCTTAATCAGGAGAATATGAAATACGTCTGGGTTCTGAATAATGATACTGAAGTGGATAAAGAGGCTTTAACTCATTTAATTAGTAAATGTGATTCAGATAAAAGTATAGGGATTTGCGGTTCTCGTTTAGTCTATTTTGCCGACAGAGAGATGCAGCAAGGACTAGGTGGGGTGCATAACAAATGGTTATGCACTACAAAAAATTATGAAATGGGAAGATTAGTTTCCAAAAAATATGATGATGAAGTCATTAGTAATGATATAGATTATATAATTGGCGCATCGATGTTTTTCTCTAGAGAATGTTTGGAAACAGTTGGATTGATGAATGAAGAATATTTTTTATACTATGAAGAGTTAGATATTTGCCTCAGAGCAAAAGCAAAGAACTTTAAATTAGGTATTTGCTCAGAAAGTTTGGTTTATCATAAAATAGGTGCAAGTACTGATGGGGGAAAGAGCATGATGGCTGATCTTTGCTCAATAAAAAATAGGCTGGTCATTACAGAAAGGTTTTATCCCCAATATTATTGGACGGTATGGTTGTCACTTTTTGTTGTAGCATTTAACCGTGCTAGAAGAGGTGAGTTTAATAAGATGAAAAGATGTTTGAATGTTATGTTTAACTTCAAACGAAACAAAGGTAGCAAATGCCATTAGAATATGCACTTAATCATGGTGTTAATAAATCTATAGTTTGATATGTTATTAAAGGGTATTTAATGAAAGTGGCTTTTTTATCTGCTTATGATCCACTATCTACATCCAGTTGGTCTGGCACACCTTATTATATGCTAAAGGCATTATCGAAGAGAAATATTTCCATTGAAATATTAGGACCGGTAAATAGCTATATGATATACATGTTAAAAGTATATAAATTAATATTAAGGTGTTTCGGAAAAGAATATGATTATAGTCATTCGAAGTTGCTTTCCAGGTATTACGGTAGAATATTCGGTAGGAAATTAAAAAAAATTGATGGTTTGGATTTTATTATCGCACCTGCAGGTTCCTCACAAATTGCTTTTTTAAAAACAACCATACCAATAATATATCTATCGGATACAACATATGATCAATTAAAAAGCTATTATCCGAATTTAAATAAAAAAACAATTATAAATGATGAGGATGCAAGTTTAATCGAACGCAAGGCTATTGAAAAAGCAACAGTAGTATCTTTCCCATCTAAATGGGCAATGGATTTTTGCAGGAATTATTACAGATTAGATTTTGATAAATTAGTTGAAATACCATGGGGGGCTAATTTATTTGATGATATTCACTTTGCTAATAAAAATATAATTCAAAAGAATAGTTATACTTGTCTTTTCTTGGGAGTTGATTGGGAAAGAAAAGGTGGGAAAACAGCCTTGAAAGCAATTGAATATGTAAGGCAGTTATATGGGATCGATGTTAGACTAAAAATTTGTGGATGTACTCCGAATCAAAAGATTTTACCTACTTGGGTTGAATTAATTGATAAAGTAGATAAAAATAACGTTGACGAATATCAGAAATTCATCGATGTGTTATCTAACGCTGATATACTTCTTTTACCAACCATTGCTGAATGTTATGGAATGGTATTTTGTGAAGCTGCTGCTTTTGGATTGCCTGTTGTCGCTACAGATACAGGTGGAGTCAGTTCTATAGTTATCAACGAAAGGACGGGGATATTAATTAAAGACCCGTTAGACTATAAGCACTTTGGAAATGCAATTCATAAAATAATTAGTTCCGTAGAGACTTATCAAAACTACTCCCAAAACGCAAGAATTAGATATAATAATATATTGCATTGGGACAATTGGGCTAAAAAGATAATTGAGATTATGTATGAGCATAAGAATAGAAGAATCAAATAGCACAAAAAGAATTATATGTTTATTTATACTTTTTCTTGTTTTCCCTGATTTTTTGTTTTATACATTAGGGGTTGATAATTTTAGCATTTCAACGATAATCTCAATTACATTGCTTTTTGTTTTTTTAAGAGCTAAAAATATTTGCAAAGATAATTTTCTAATAATAGTAGCGTTATTCATATTGTTGTGTTTTAACTGTTTGTTAAGTATGCTATTTAATATTGAACAGGCTTTAACATTTAAAGTTGTACTTTCAATATATAGCATCTTAATAATGGCATACGTCTCCTCTTGTTATGCACAGACGTTGTGGTTATGTTCTGAAGAAATACTTAAGAGATCCGTCTTTTATTTGTTCGCATTTCTTTGCCTTATTGGCATTATAAGTATTCTTTTACAGAAGACTGAGATTATACATGATAAAAGTATGATTCTTTTTCCTGAACCATCAGCATTTGCATTGGTTTTTATACCTATCTTTTCATTTTGTTTATACTATACAAGAGGGGGGGGGCTACTATTGCTCTATATATTATCTTTGGGTATTGCGTTAGGTATCCAGAATTTAACAATGTTGGTAGGCATTGTGATTAGTGTTTTTGTGATGAAAAAAATAACTATAAGGCAAACTATTGTTATACTTTTGGGGGCATGGATTTTTTCCATGATATTAAGTGATTTAGACATTTCTTACTATACATCGCGGCTTGATTTTAAAAATACTACGAACCTATCAGTGCTTGTATATCTTTCAGGAATTGAAAGAGCTTTCTTGAATTTTATTACAAGTTATGGTCTTGGTATTGGTTTTCAACAAATGGGAGTGAATGGGGAGATAGGAATATATCAACAAATTTTAGCTGAACTTGATGCCCCTATGTTAAATATATACGATGGCTCATTTATTTCTTCTAAGTTAATATCTGAGTTTGGGGTTATTGGTGCATTAATGTGTATTTTCTATTTTTTTTATTTTTCCCGATTTTATCTGCGTTTCAAAAAAAGTAAGAGATATTCACCGCAGTATATTTTAGCATATAGCTTCTACATGTGTTTCTTCATCCCTCTTTTTATACGTGGTGCTGGTTATATAAACCCCTATGTGTTTATGTTATTTTCATCAATATTTTTGTGCAAATATCACGCTAAAAATATCTTGATGAAATCTAATGTCCAGATAGCTATATAATAGTAGATTATATTATCATTATCACGTAAATTACATATTAATAGCATATATGATAACTAGGACATAAATAATGTGCATTAAAAAAAAACTTAAGTTAATTAAACGATATGGCCTTTATGGTGGTCTTAGGCTTCTTAAAGATATATTCTTAACAAAATTTTTATTTTGTTCAAATGTTAGGATTATTAGATTTCCATGTTATATTAGAAAAGATGGAAGTGTTAGTTTTGGAAAAGGTTTTACATCAGGTGTAGGATTACGAGTTGATGCATTTATGGATGCCGTAGTTTCCATTGGAGAAAATGTTCAAATTAATGACTATGTTCACATCGCGGCTATTAATAATGTCATTATTGGTAGAGATACATTAATAGCAAGTAAAGTATTTATTAGTGATCATAATCATGGTATTTTTTCTAAATCCGATATCCATAGTTCACCAACTATTATTCCTTCGTCTAGGCCCCTTGAATCTGCACCTGTGTATATTGGAGAGCGTGTGTGGATTGGCGAAAATGTGACAATATTACCAGGTGCGTGTATAGGTAATGGTGTAGTTATTGGCGCAAACAGTGTTGTTCGTGGTGAGATTCCTAATAATGTGATCATTGCTGGTGTTCCAGCTAAAATTGTTAAAAAATATAACTATGAGCGTATGCAATGGGAAAGAATATAGTTGTAATATCGGCTGTTAATTTTACAACCGGAGGCCCCTTTACCGTACTAAAAAATGTGCTTACAGCAACTAAAGATAGAGCCGAATGTAAATTTATTGCACTGGTTCATAGCTCTGCTGAACTAATGGAATTATTTCCGTGGGTTGAATTTATAGAGTATCCAGAAGTCAAGTCTTCGTGGGTTAAAAGATTATATTTCGAATATATAACTTGCAATAGATTATCTAAGGTGATTAAGGCAACTCATTGGGTATGCTTACATGATATTACAGCAAATGTTAGTGTACCCTATAGATTTGTTTATTGCCACAATCCTGCACCGTTCTATAAATATTTAAGCTATCGAGATATTATAGGAGAACCTAAATTTTATCTTTTTTATCTTTTTTATGGGCTTTTATACAATATCAATATAAAAAAGAACACAGCAGTTTTTGTTCAGCAGCAGTGGCTAAAAAAAGAATTCGAAAAAAAATATAAGTTAAAGAATGTTGTTGTTAGTCGCCCTGAAGATATTTGCCCTTTTGAAAGTGATGGTTTGGTAAGAAATAATAATAAAAAGGATGTGAGGATATTTTACCCAGCAGTGCCCCGTATATTTAAAAACTTTGAAGTTATCATACGTGCTGCACAAATATTACAAGATAAAAATATTCATTTTTATCTTACTTTTGATGGTACTGAAAATAAGTATGCAAAAGAATATATAAATTAGCTTCCGAACTGAAAAATGTACATTTCCTCGGTTACCTTAATGCAACCGAGATGGTTAACTTTTATCAAGATTCAGATATTATTTGTTTCCCATCGAAACTAGAAACGTGGGGATTACCATTATCAGAAGCTAAAACATACAAAAAATGGATATTTGCGGCAGACTTACCTTATGCTCATGAAGTTTTATATAACTATTCAAAAACTAGATATTTTCCATTTGACGATGAGAAAATACTTGTTCGCTACATATTAGAGTACACAAGTAAAAATATGCATGAAGATATAAAAAATAGTAGGGTGAATTTTAATAATGATGCATTGACTGGTTTTGAACAGTTTATTGAATATATCCTCAAGGGGAACTGACGTGGTTTATATTATAATCGTTTCACATGGCCATGATGACTATATAGAAAATCTTTTATTAAATTTAAAGTTGCCCTCTGGAAGATTTAAAATAATAGTTCGTGATAACAAAAGTTCAATGGTTTTAAAAAAAACATGCGAAAAAAATTGCGTAACCTATTTGCATGGAGGGCAATATGGATTTGGACATAATAATAACATAGCAGTGTCATATATAATTAATAACTTCATGATTATGAATAATGATTATTTTCTCTTTCTTAACCCCGATGTATTCATAACCAGTGAAAGTTTGATTAATTATGTTGATTATATAATTAGTAATGATTATAAGTTTAGCACATTATGTCTTTATCGAGATTTTACTAAAAGCAAACATGATTATTCAATACGGAGTTTTCCAACTTTATATGATTTTCTTTGTTCTTTTTTATTGGGGGTGAATAAAAGTAAAATTAAGAAGGAAAATATACTTTCTGATACTGTAGTTGATTGGTGTGCTGGCTCATTTATGCTTATTCATGCTTTAAGTTTCTTAAATGTGAATGGTTTTGATCAAAAATATTTTATGTATTGTGAAGATATTGACCTTTGTATGCGTTTAAAATTAAGTGGAGTAGATCTTTACTATACTCCCCATTTTGATGCTATTCATTATGCGCAGCATGAAAATAGAAGAATATTTACTAAAGCATTTCGATGGCATATAAGGAGTATTACGCGCTACATATTACGGAAACCAATTCTTTCTTATAAAAACTATAGAAAAATTACATCCGAACTGGTAAAGTGA Detoxified EPAGSGGGDQNATGSGGGKLAEEAFDLWNECAKACVLDLKDGVRSSRM 13 proteinSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTI comprising 4RLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIH optimized N-ELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQP glycosylationTLAISHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNY sequencesLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKDNNNSTPTVISHRLHFPEGGSLAALTAHQACHLPLEAFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVAKDQNRTKGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRWSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRVTILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQ PGKPPREDLKLGSGGGDQNATN-glycosylation Asn-X-Ser(Thr), wherein X can by any amino 14 consensusacid except Pro sequence N-glycosylationAsp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z 15 consensusare independently selected from any natural sequenceamino acid except Pro

The embodiments described herein are intended to be merely exemplary,and those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, numerous equivalents to thespecific procedures described herein. All such equivalents areconsidered to be within the scope of the present invention and arecovered by the following claims.

All references (including patent applications, patents, andpublications) cited herein are incorporated herein by reference in theirentirety and for all purposes to the same extent as if each individualpublication or patent or patent application was specifically andindividually indicated to be incorporated by reference in its entiretyfor all purposes.

7. EMBODIMENTS Embodiments 1

1. A prokaryotic host cell comprising:

-   -   a. rfb(upec138) gene cluster (SEQ ID NO:12), rfb(upec163) gene        cluster, or rfb(upec177) gene cluster;    -   b. a nucleotide sequence encoding an oligosaccharyl transferase;        and    -   c. a nucleotide sequence encoding a carrier protein comprising a        consensus sequence Asn-X-Ser(Thr), wherein X can be any amino        acid except Pro (SEQ ID NO:14).

1.-16. (canceled)
 17. A recombinant prokaryotic host cell for producingan N-glycosylated carrier protein, comprising: a. a nucleotide sequenceencoding: i. dTDP-Glucose 4,6-dehydratase; ii. dTDP-6-Deoxy-D-glucose3,5-epimerase; iii. Glucose-1-phosphate thymidylyltransferase; iv.dTDP-4-dehydrorhamnose 3,5-epimerase; v. O antigen flippase; vi.dTDP-Rha:Glc-Rha(Ac)-GlcNAc-UPP α-1,3-rhamnosyltransferase; vii.UDP-Glc:Glc-Rha(Ac)-GlcNAc-UPP α-1,6-glucosyltransferase; viii. Oantigen polymerase; ix. O-acetyl transferase; x. UDP-Glc:Rha-GIcNAc-UPPα-1,3-glucosyltransferase; and xi. dTDP-Rha: GlcNAc-UPPα-1,3-rhamnosyltransferase; b. a nucleotide sequence encoding anoligosaccharyl transferase; and c. a nucleotide sequence encoding acarrier protein comprising a consensus sequenceAsp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selectedfrom any natural amino acid except Pro (SEQ ID NO:15).
 18. The host cellof claim 17, wherein at least one of the waaL gene, gtrA gene, gtrBgene, gtrS gene, or rfb cluster is deleted from or functionallyinactivated in the genome of the host cell.
 19. The host cell of claim17, wherein the carrier protein is selected from the group consisting ofdetoxified Exotoxin A of P. aeruginosa (EPA), CRM197, maltose bindingprotein (MBP), Diphtheria toxoid, Tetanus toxoid, detoxified hemolysin Aof S. aureus, clumping factor A, clumping factor B, E. coli FimH, E.coli FimHC, E. coli heat labile enterotoxin, detoxified variants of E.coli heat labile enterotoxin, Cholera toxin B subunit (CTB), choleratoxin, detoxified variants of cholera toxin, E. coli Sat protein, thepassenger domain of E. coli Sat protein, Streptococcus pneumoniaePneumolysin and detoxified variants thereof, C. jejuni AcrA, and C.jejuni natural glycoproteins.
 20. The host cell of claim 17, whereinsaid host cell is an E. coli host cell.
 21. A method of making anN-glycosylated carrier protein, said method comprising: a. culturing thehost cell of claim 17 under conditions for producing the N-glycosylatedcarrier protein; and b. purifying the N-glycosylated carrier protein.22. The method of claim 21, wherein the host cell is E. coli.
 23. Themethod of claim 21, wherein the carrier protein is detoxified Exotoxin Aof P. aeruginosa (EPA).
 24. The method of claim 21, wherein theN-glycosylated carrier protein comprises an E coli O25B antigen havingthe structure of Formula O25B′:

wherein n is an integer of 1 to
 30. 25. The method of claim 21, whereinthe nucleotide sequence encoding the carrier protein further comprises anucleotide sequence encoding a signal sequence for targeting the carrierprotein into the periplasmic space of the host cell.
 26. A method ofmaking an N-glycosylated carrier protein that comprises an E. coli O25Bantigen, the method comprising: a. culturing a recombinant prokaryotichost cell under conditions for producing the N-glycosylated carrierprotein, wherein the prokaryotic host cell comprises: i. a nucleotidesequence comprising a rfb gene cluster encoding the E. coli O25Bantigen; ii. a nucleotide sequence encoding an oligosaccharyltransferase; and iii. a nucleotide sequence encoding a carrier proteincomprising a consensus sequence Asn-X-Ser(Thr), wherein X is any aminoacid except Pro (SEQ ID NO:14); and b. purifying the N-glycosylatedcarrier protein that comprises the E. coli O25B antigen.
 27. The methodof claim 26, wherein the host cell is E. coli.
 28. The method of claim26, wherein the consensus sequence comprises Asp(Glu)-X-Asn-Z-Ser(Thr),wherein X and Z are independently selected from any natural amino acidexcept Pro (SEQ ID NO:15).
 29. The method of claim 26, wherein the O25Bantigen comprises the formula O25B′:


30. The method of claim 26, wherein the carrier protein is selected fromthe group consisting of detoxified Exotoxin A of P. aeruginosa (EPA),CRM197, maltose binding protein (MBP), Diphtheria toxoid, Tetanustoxoid, detoxified hemolysin A of S. aureus, clumping factor A, clumpingfactor B, E. coli FimH, E. coli FimHC, E. coli heat labile enterotoxin,detoxified variants of E. coli heat labile enterotoxin, Cholera toxin Bsubunit (CTB), cholera toxin, detoxified variants of cholera toxin, E.coli Sat protein, the passenger domain of E. coli Sat protein,Streptococcus pneumoniae Pneumolysin and detoxified variants thereof, C.jejuni AcrA, and C. jejuni natural glycoproteins.
 31. The method ofclaim 26, wherein the carrier protein is detoxified Exotoxin A of P.aeruginosa (EPA).
 32. The method of claim 26, wherein the nucleotidesequence encoding the carrier protein further comprises a nucleotidesequence encoding a signal sequence for targeting the carrier proteininto the periplasmic space of the host cell.
 33. The method of claim 31,wherein the host cell is E. coli.
 34. The method of claim 33, whereinthe consensus sequence comprises Asp(Glu)-X-Asn-Z-Ser(Thr), wherein Xand Z are independently selected from any natural amino acid except Pro(SEQ ID NO:15).
 35. The method of claim 34, wherein the nucleotidesequence encoding the carrier protein further comprises a nucleotidesequence encoding a signal sequence for targeting the carrier proteininto the periplasmic space of the host cell.
 36. The method of claim 35,wherein the O25B antigen comprises the formula O25B′: